Peptide Aptamers that Bind to the Rep Proteins of ssDNA Viruses

ABSTRACT

Polypeptides and fusion proteins that bind to eukaryotic viruses, in particular, eukaryotic single-stranded DNA (ssDNA) viruses are provided. The polypeptides and fusion proteins bind to the replication initiation proteins (Rep) of ssDNA viruses and optionally inhibit viral replication and/or viral infection. The virus can be a plant pathogen or animal pathogen. Consensus sequences used to identify polypeptides that bind to eukaryotic viruses are also provided.

RELATED APPLICATION DATA

The present application is a divisional of U.S. patent application Ser.No. 11/995,973 (now allowed), which is a 35 U.S.C. §371 national phaseapplication of PCT Application No. PCT/US2006/030941, filed Aug. 4,2006, which claims the benefit of U.S. provisional application Ser. No.60/705,426, filed Aug. 4, 2005, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to products and methods for detectingviral infections and inhibiting viral replication and products resultingtherefrom.

BACKGROUND OF THE INVENTION

Single-stranded DNA (ssDNA) viruses cause severe disease problems inplants and animals (Moffat (1999) Science 286:1835). Geminiviruses andnanoviruses infect many important crops worldwide, such as cassava,bean, pepper, tomato, sugar beet, cotton and maize (Brown and Bird(1992) Plant Disease 7:220-225; Czosnek and Laterrot (1997) Arch. Virol.142:1391-1406; Lotrakul, et al. (1998) Plant Dis. 82:1253-1257; Zhou, etal. (1997) J. Gen. Virol. 78:2101-2111; Mansoor, et al. (1999) Virology259:190-199; Polston, et al. (1999) Plant Dis. 83:984-988). Circovirusescause significant disease losses among livestock and poultry (Allan, etal. (1998) J. Vet. Diagn. Invest. 10:3-10; Bassami, et al. (1998)Virology 249:453-9; Nayar, et al. (1999) Can. Vet. J. 40:277-8). A humancircovirus in Hepatitis C patients has also been identified (Miyata, etal. (1999) J. Virol. 73:3582-3586; Mushahwar, et al. (1999) Proc. Natl.Acad. Sci. USA. 96:3177-3182). Even though these viruses have diversehost ranges and cause different diseases, they are highly related toeach other.

Geminiviruses, nanoviruses, and circoviruses amplify their circularssDNA genomes via a rolling circle mechanism through the combined actionof a single viral protein, Rep, and the host DNA replication machinery(Laufs, et al. (1995) Biochimie 77:765-773; Mankertz, et al. (1997) J.Virol. 71:2562-2566; Katul, et al. (1998) J. Gen. Virol. 79:3101-3109;Mankertz, et al. (1998) J. Gen. Virol. 79:381-384; Hanley-Bowdoin, etal. (1999) Crit. Rev. Plant Sci. 18:71-106). Rep initiates plus-strandDNA synthesis by cleaving the viral origin within a hairpin structure atan invariant sequence, acts as a DNA ligase to terminate rolling circlereplication, and hydrolyzes ATP. Because of the functional conservation,Rep proteins from all three ssDNA virus families are highly homologous.

The Geminiviridae family is classified into four genera based on genomestructure, insect vector and type of host (Rybicki 1994; Briddon,Bedford et al. 1996). The four genera infect a broad range of plants andcause significant crop losses worldwide (Brown and Bird 1992; Brown1994; Rybicki and Pietersen 1999; Morales and Anderson 2001; Mansoor,Briddon et al. 2003). All geminiviruses are characterized by twinicosahedral capsids (Zhang, Olson et al. 2001; Bottcher, Unseld et al.2004) and single-stranded DNA (ssDNA) genomes that replicate throughdouble-stranded DNA (dsDNA) intermediates (Hanley-Bowdoin, Settlage etal. 1999).

Geminiviruses replicate their small, circular DNA genomes using acombination of rolling circle and recombination-mediated replication(Gutierrez 1999; Jeske, Lutgemeier et al. 2001). They encode theproteins required for initiation of replication, Geminivirus ReplicationInitiation Protein (Rep), and depend on host polymerases for DNAsynthesis (Gutierrez 2000; Hanley-Bowdoin, Settlage et al. 2004). Muchof our knowledge of geminivirus replication comes from studies of TGMV,a typical begomovirus with a bipartite genome. Two of the seven proteinsencoded by TGMV are involved in viral replication. AL1 is required forviral replication (Elmer, Brand et al. 1988; Hanley-Bowdoin, Elmer etal. 1990), whereas AL3 is an accessory factor that enhances viral DNAaccumulation (Sunter, Hartitz et al. 1990). The AL1 protein showsconservation across all four genera. Different nomenclatures have beenused to designate AL1, which is also known as Rep, AC1 or C1. As usedherein, the Rep designation is employed because it is applicable to allgeminiviruses.

Rep is a multifunctional protein that mediates both virus-specificrecognition of its cognate origin (Fontes, Eagle et al. 1994) andtranscriptional repression (Eagle, Orozco et al. 1994; Eagle andHanley-Bowdoin 1997). Rep initiates and terminates (+) strand DNAsynthesis within a conserved hairpin motif (Heyraud-Nitschke, Schumacheret al, 1995; Laufs, Traut et al. 1995; Orozco and Hanley-Bowdoin 1996).It also induces the accumulation of host replication factors in infectedcells (Nagar, Pedersen et al. 1995). Rep binds to dsDNA at a repeatedsequence in the origin (Fontes, Eagle et al. 1994; Fontes, Gladfelter etal. 1994), cleaves and ligates DNA within an invariant sequence of ahairpin loop (Laufs, Jupin et al. 1995; Orozco and Hanley-Bowdoin 1996),and is thought to unwind viral DNA in an ATP-dependent manner(Gorbalenya and Koonin 1993; Pant, Gupta et al. 2001). Rep interactswith itself and AL3 (Settlage, Miller et al. 1996). It binds to severalhost factors involved in DNA transactions, including the replicativeclamp PCNA (Castillo, Collinet et al. 2003), the clamp loader RFC(Luque, Sanz-Burgos et al. 2002), histone H3 and a mitotic kinesin (Kongand Hanley-Bowdoin 2002). Rep also interacts with host regulatoryfactors, including the retinoblastoma protein (pRBR) which modulates thea cell cycle and differentiation (Xie, Suarezlopez et al. 1995; Grafi,Burnett et al. 1996; Ach, Durfee et al. 1997), a novel protein kinase(GRIK) associated with leaf development (Kong and Hanley-Bowdoin 2002),and Ubc9—a component of the sumoylation pathway (Castillo, Kong et al.2004).

The functional domains of Rep have been mapped by deletion andmutational studies (FIG. 1). The N-terminal half of Rep containsoverlapping domains for DNA cleavage/ligation, DNA binding, and proteininteractions (Orozco, Miller et al. 1997; Orozco and Hanley-Bowdoin1998). NMR spectroscopy revealed that the overlap-ping DNAbinding/cleavage domains contain a β-sheet cluster that resemble othernucleic acid binding proteins (Campos-Olivas, Louis et al. 2002). Thecharacterized Rep protein interactions fall into two classes—proteinsthat bind between amino acids 101-180 (Kong, Orozco et al. 2000;Settlage, Miller et al. 2001) and those that bind between amino acids134-180. (Orozco, Kong et al. 2000; Kong and Hanley-Bowdoin 2002). Theputative DNA helicase domain is in the C-terminus (Gorbalenya and Koonin1993; Pant, Gupta et al. 2001).

Rep contains several conserved amino acid and structural motifs (FIG.1). Motifs I, II and III are characteristic of rolling circle initiators(Ilyina and Koonin 1992; Koonin and Ilyina 1992). Motif I (FLTY) is adeterminant of dsDNA binding specificity (Chatterji, Chatterji et al.2000; Arguello-Astorga and Ruiz-Medrano 2001). Motif II (HLH) is a metalbinding site that may impact protein conformation and/or catalysis.Motif III (YxxKD/E) is the catalytic site for DNA cleavage with thehydroxyl group of the Y residue forming a covalent bond with the 5′ endof the cleaved DNA strand (Laufs, Traut et al. 1995). The aromatic ringof the Y residue plays a role in dsDNA binding (Orozco andHanley-Bowdoin 1998). The three motifs are exposed and in closeproximity on the β-sheet surface in the Rep N-terminus (Campos-Olivas,Louis et al. 2002). Other conserved motifs include a sequence of nearidentity and unknown function immediately C-terminal of Motif III (Kong,Orozco et al. 2000), a helix-loop-helix motif that mediates pRBR binding(Arguello-Astorga, Lopez-Ochoa et al. 2004), and a NTP binding consensus(Walker, Saraste et al. 1982).

A variety of strategies have been applied to geminivirus resistance,including conventional breeding and transgenic approaches. Conventionalbreeding has been confounded by the limited sources of naturalresistance, the multigenic nature of the resistance traits, and the timerequired for a breeding program (Miklas, Johnson et al. 1996; Pessoni,Zimmermann et al. 1997; Velez, Bassett et al. 1998; Welz, Schechert atal. 1998; Kyetere, Ming et al. 1999). TYLCV resistance genes have beenintrogressed from a wild Lycopersicon species (Pilowsky and Cohen 1990;Lapidot, Friedmann et al. 1997; Friedmann, Lapidot et al. 1998; Vidayskyand Czosnk 1998). This resistance is often unsatisfactory due to linkagewith poor fruit quality, complex inheritance patterns, and thedifficulty of transfer to commercial cultivars. Most conventionalresistances collapse under early or severe infection pressure (Lapidotand Friedmann 2002). There is also evidence that host resistance genesare not equally effective against different geminiviruses (Pernet,Hoisington et al. 1999; Pernet, Hoisington et al. 1999), and many hostgenes only confer tolerance (Gilreath, Shuler at al. 2001; Lapidot,Friedmann et al. 2001; Gomez, Pinon et al. 2004). Tolerant plants, whichsupport viral replication—albeit at lower levels, can serve asreservoirs for mutant and recombinant viruses that have the potential toovercome resistance.

Several transgenic strategies based on pathogen-derived resistance havealso been tested. There is one report of transgenic tomatoes thatcontain a mutant begomovirus coat protein gene and display tolerance(Kunik, Salomon et al. 1994), but this result has not been reproduced byother researchers using wild type viral sequences (Azzam, Diaz et al.1996; Sinisterra, Polston et al. 1999). Instead, expression ofgeminivirus sequences frequently results in the production of functionalproteins that typically complement defective viruses or cause symptoms(Hanley-Bowdoin, Elmer et al. 1989; Hayes and Buck 1989; Hanley-Bowdoin,Elmer et al. 1990; Pascal, Goodlove et al. 1993; Latham, Saunders et al.1997; Krake, Rezaian et al. 1998; Guevara-Gonzalez, Ramos at al. 1999;Hou, Sanders at al. 2000:Sunter, 2001 #7731). The reduced sensitivity topathogen-derived resistance may reflect the lack of an RNA genomic formand the ability of geminiviruses to modulate host gene silencing(Ratcliff, Harrison et al. 1997; Voinnet, Pinto et al. 1999; Covey andAlKaff 2000; Noris, Lucioli et al. 2004; Vanitharani, Chellappan et al.2004). Antisense RNA and defective-interfering replicon strategies havealso been of limited success (Stanley, Fischmuth et al. 1990; Day,Bejarano et al. 1991; Frischmuth and Stanley 1994; Aragao, Ribeiro etal. 1998; Asad, Haris et al. 2003). Recent reports suggested that RNAiconstructs can confer strong resistance, but this strategy is limited tohomologous (or very closely related) geminiviruses (Pooggin, Shivaprasadet al. 2003; Pooggin and Hohn 2004). Transgenic plants that induciblyexpress dianthin upon geminivirus infection also display resistance(Hong, Saunders et al. 1996), but the safety of a toxicribosome-inactivating protein has not been established. Expression ofmutant begomovirus movement proteins in transgenic plants also resultedin resistance, but the phenotype is variable possibly because of theability of the mutant proteins to confer symptoms in the absence ofinfection (Pascal, Goodbye et al. 1993; Duan, Powell et al. 1997; Duan,Powell et al. 1997; Hou, Sanders et al. 2000).

Unlike the strategies described above, Rep mutants have proven effectiveat interfering with geminivirus replication in cultured cells. Mutationsin Motif III, the ATP binding site and the oligomerization domain(FIG. 1) interfere with virus replication in transient assays (Hansonand Maxwell 1999; Orozco, Kong et al. 2000; Chatterji, Beachy et al.2001). However, plants that stably produce the Rep protein displayde-velopmental defects (Brunetti, Tavazza et al. 1997; Brunetti, Tavazzaet al. 2001), and expression is selected against during meiosis. ThepRBR protein is required for gametogenesis (Ebel, Mariconti et al.2004), suggesting that the Rep expression problem reflects itsinteraction with pRBR. Recent experiments showed that inclusion of apRBR binding mutation in an interfering Rep transgene results in stableexpression through at least 3 generations. Because Rep is highlyspecific for its cognate viral origin (Fontes, Gladfelter et al. 1994;Chatterji, Chatterji et al. 2000), the same plants were designed to alsoexpress a mutant AL3 in an effort to the broaden resistance. (AL3functions in virus-nonspecific manner to enhance viral accumulation(Santer, Stenger et al. 1994; Sung and Coutts 1995)). The plantscoexpressing the mutant Rep and AL3 proteins are immune to infection bythe ho-mologous virus through at least three generations. It is not yetknown if they are resistant to unrelated begomoviruses. In contrast,other studies showed that infection with a homologous virus can lead toRep transgene silencing. It is desirable to develop alternativeresistance strategies.

Peptide aptamers resemble single chain antibodies, but because of theirin vivo selection, are more likely to be stably expressed and correctlyfolded and to interact with their targets in an intracellular context(Crawford, Woodman et al. 2003). If an aptamer binds to residuescritical for function, it can inactivate its target and interfere withcellular processes. For example, an aptamer that binds to the activesite of the cell cycle regulator, cdk2, was isolated by screening acombinatorial peptide library in yeast dihybrid assays (Colas, Cohen etal. 1996). The aptamer blocks cdk2/cyclin E kinase activity in vitroand, when expressed in vivo, retards cell division (Cohen, Colas et al.1998). An aptamer that interacts with the dimerization domain of cellcycle-associated transcription factor, E2F, also interferes with cellcycle progression in animal cells (Fabbrizio, LeCam et al. 1999).Aptamers have also been expressed in flies to study the specific rolesof cdk1 and cdk2 during Drosophila organogenesis (Kolonin and Finley1998). They have been used to distinguish between and selectivelyinactivate allelic variants of Ras and to inhibit Rho GTP exchangefactors (Schmidt, Diriong et al. 2002; Xu and Luo 2002; Kurtz, Espositoet al. 2003) as well as interfere with the EGF signaling pathway, bybinding to the downstream transcription factor Stat3 (Buerger,Nagel-Wolfrum et al. 2003; Nagel-Wolfrum, Buerger et al. 2004).

Peptide aptamers are especially well suited for targeting noncellularfactors like viral proteins. An aptamer that binds to the hepatitis Bvirus core protein and inhibits viral capsid formation and replicationhas strong antiviral activity in liver cells (Butz, Denk et al. 2001).Aptamers that target the E6 or E7 proteins of human papillomavirus andblock their anti-apoptotic activities result in specific elimination ofHPV-positive cancer cells (Butz, Denk et al. 2000; Nauenburg, Zwerschkeet al. 2001).

The present inventors have found that expression of aptamers that targetessential, conserved Rep motifs can interfere with viral replication andconfer broad resistance against geminivirus infection.

SUMMARY OF THE INVENTION

Single-stranded DNA (ssDNA) viruses cause severe disease problems inplants and animals. Geminiviruses and nanoviruses infect many importantplant crops worldwide, whereas circoviruses cause significant diseaselosses among livestock and poultry. Even though these viruses havediverse host ranges and cause different diseases, their replicationinitiation proteins (Rep) are highly related to each other. The presentinvention can be used to develop a broad-based resistance strategydirected to eukaryotic viruses, in particular, eukaryotic ssDNA viruses.

A first aspect of the invention provides a polypeptide comprising,consisting essentially of or consisting of an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ IDNO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124,SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ IDNO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO: 137, SEQ ID NO:138,SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO:143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ IDNO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166,SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ IDNO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180,SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:169, SEQID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ IDNO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, or any combination thereof;

A further aspect of the invention provides a fusion protein comprisingthe foregoing polypeptides.

Additional aspects of the invention provide an isolated nucleic acidcomprising a nucleotide sequence encoding a polypeptide of theinvention.

A further aspect of the present invention provides a vector or a cellcomprising an isolated nucleic acid comprising a nucleotide sequenceencoding the polypeptide comprising an amino acid sequence as recitedabove.

Further aspects of the present invention provide a transgenic plantcomprising transformed plant cells, the transformed plant cellscomprising the isolated nucleic, acids of the invention. A plurality oftransgenic plants, such as a crop of transgenic plants, comprising thetransformed plant cells described herein is also provided.

A further aspect of the invention provides a transgenic plant havingincreased resistance to a geminivirus infection, a viral variant thereofand mixed infections. A plurality of transgenic plants, such as a cropof transgenic plants, having increased resistance to a geminivirusinfection, a viral variant thereof and mixed infections is alsoprovided.

Also provided are methods of making transgenic plants having increasedresistance to a virus infection, said method comprising providing aplant cell capable of regeneration, transforming the plant cell with anisolated nucleic acid comprising an isolated nucleic acid as describedherein, and regenerating a transgenic plant from said transformed plantcell, wherein expression of the isolated nucleic acid to produce thepolypeptide increases resistance of the transgenic plant to infection bya virus. Additional methods of making transgenic plants having increasedresistance to a virus infection comprise introducing an isolated nucleicacid, as recited herein, into a cell to produce a transgenic plant,wherein expression of the isolated nucleic acid to produce thepolypeptide increases resistance of the transgenic plant to infection bya virus.

Another aspect of the present invention provides a method of inhibitingviral replication in a plant cell comprising introducing an isolatednucleic acid, as recited herein, into the plant cell in an amounteffective to inhibit virus replication.

The present invention further provides methods of detecting a viralinfection, comprising: (a) contacting a sample with a polypeptide asrecited above or a fusion protein as recited herein; and (b) detectingthe presence or absence of binding between the polypeptide or fusionprotein and a target, wherein the binding of the polypeptide or fusionprotein to the target in the sample indicates the presence of a virus.

A further aspect of the present invention provides polypeptidesidentified through a method comprising identifying polypeptides thatcorrespond to consensus peptide sequences derived from statisticalanalysis of a library of peptide sequences.

The invention further provides polypeptides that target a ssDNA virusreplication initiation protein and interfere with the function of thereplication initiation protein in vivo.

Further aspects of the invention provide a method of treating a viralinfection in a subject in need thereof comprising administering apolypeptide according to embodiments of the present invention to thesubject. The polypeptide can be formulated in a suitable pharmaceuticalor agricultural carrier.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts functional domains and motifs of the Rep protein. MotifsI, II and III are associated with rolling circle replication initiatorproteins. The solid box is the conserved element of unknown function.The conserved helix-loop-helix motif marked by the oval provides theprimary pRBR contacts. The box marked “ATP” is the NTP binding site ofthe putative DNA helicase domain. The limits of the functional domainsfor DNA cleavage/ligation, DNA binding, and known protein interactionsites are indicated. The PCNA binding site has been localized to the RepN-terminus but has not been finely mapped. The RFC and H3 binding siteshave not been mapped. The numbers at the top indicate amino acidpositions in TGMV Rep.

FIG. 2 presents baits for aptamer screens. FIG. 2A presents diagrams ofthe TGMV AU coding regions (TAL1₁₋₃₅₂ and TAL1₁₋₁₃₀) cloned downstreamof the LexA DBD. Motifs I, II and III associated with rolling circlereplication initiator proteins are marked by the black boxes and theirconsensus are shown (Ilyina, T. V., and E. V. Koonin 1992. Conservedsequence motifs in the initiator proteins for rolling circle DNAreplication encoded by diverse replicons from eubacteria, eucaryotes andarchaebacteria. Nucleic Acids Res. 20:3279-3285; Koonin, E. V., and T.V. Ilyina. 1992. Geminivirus replication proteins are related toprokaryotic plasmid rolling circle DNA replication initiator proteins.J. Gen. Virol. 73:2763-2766). The oval corresponds to a conservedhelix-loop-helix motif and the grey box is the ATP binding motif. InFIG. 2B, baits were tested for oligomerization activity using thepositive (AD:TAL1₁₋₃₅₂) and the negative (AD:Jun) prey controls. Theyeast transformants were [1] TAL1₁₋₃₅₂+AD:TAL1₁₋₃₅₂, [2]TAL1₁₋₃₅₂+AD:Jun, [3] TAL1₁₋₁₃₀+AD:TAL1₁₋₃₅₂, [4] TAL1₁₋₁₃₀+AD:Jun, [5]GUS+AD:TAL1₁₋₃₅₂, [6] GUS+AD:Jun, [7] CaAL1₁₋₃₄₉+AD:TAL1₁₋₃₅₂ and [8]CaAL1₁₋₃₄₉+AD:Jun. Interaction was monitored by growth on Gal-HWULmedium. Growth on Glu-HWU controlled for plasmid selection, whereas nogrowth on Glu-HWUL verified that interaction was dependent on inductionof prey plasmid expression.

FIG. 3 shows that aptamers that bind to TAL1₁₋₁₃₀ also interact withTAL1₁₋₃₅₂. The 88 plasmids recovered from the screen of the JM-1 libraryusing TAL1₁₋₁₃₀ as bait were retransformed into different bait strainsto confirm specificity of interaction. FIG. 3A presents a key forN-TrxA-peptides on the plates shown in FIGS. 3B-3D. Controls in column12 are numbered as in FIG. 2. The interaction assay was performed onGal-HWUL (B), Glu-HWUL (C) and Glu-HWU (D) media with the TAL1₁₋₁₃₀,TAL1₁₋₃₅₂ and GUS baits as indicated at the top. Peptides that interferewith replication of TGMV are boxed in FIG. 3A.

FIG. 4 shows results of replication interference assays. FIG. 4Apresents a diagram showing the input replicon cassette, the releasedTGMV A replicon, and the plant expression cassettes. The positions ofprimers (LLp1 and LLp2) used to distinguish input vector and replicatedDNA are marked. In FIG. 4B, tobacco protoplasts were cotransfected witha TGMV A replicon (pMON1565; lanes 1-4) and a plant expression cassette.Total DNA was isolated 36 h post transfection, digested with DpnI andXhoI, and analyzed on DNA gel blots using a virus-specific probe fordouble-stranded DNA accumulation (dsDNA). The expression cassettescorrespond to the trans-dominant TAL1 mutant FQ118 (pNSB866; lane 1), anempty cassette (pMON921; lane 2), aptamer FL-42 (pNSB1136; lane 3) andaptamer FL-60 (pNSB1144; lane 4). In FIG. 4C, released DNA was amplifiedfrom E. coli transfected with an AL1 mutant replicon cassette. Total DNAwas isolated from E. coli transformed with either a wild type TGMV Areplicon cassette (pMON1565; lanes 1-3) or a mutant replicon cassettecarrying an AU frame-shift mutation (pMON1679; lanes 4-6) and amplifiedusing primers LLp1 and LLp2 in (A). The methylation status of thetemplate DNAs was assessed by digestion with DpnI (lanes 2 and 5) andMboI (lanes 3 and 6). PCR products corresponding to the repliconcassette and released TGMV A DNA are marked. Markers corresponding to100-bp (lane 7) and 1-kb (lane 8) DNA ladders are shown. As shown inFIG. 4D, TGMV A replication required full length AL1 in plant cells.Tobacco protoplasts were transfected with a wild type TGMV A replicon(pMON1565; lanes 1-9) or the mutant AL1 replicon cassette (pMON1679;lanes 10-12). In lanes 1-9, plant expression cassettes corresponding toan empty cassette (pMON921; lanes 1-3), the trans-dominant AL1 mutantFQ118 (pNSB866; lane 4-6) and the TrxA-GST control (pNSB1166; lanes 7-9)were included in the transfections. Total DNA was isolated 36 h posttransfection and analyzed directly by PCR or after digestion with DpnIor MboI.

FIG. 5 shows results of studies designed to study interference with TGMVreplication for aptamers that bind to TAL1₁₋₁₃₀. The N-TrxA-peptidesselected by screening with TAL1₁₋₁₃₀ and cloned into plant expressioncassettes (Table 4) were tested in replication interference assays usingthe semi-quantitative PCR assay shown in FIG. 4D. Bands corresponding tothe replicated TGMV A DNA (1.2 Kb) and the PCR internal control (700 bp)were quantified using imageJ software (Abramoff, M. D., P. J. Magelhaes,and S. J. Ram. 2004. Image processing with ImageJ. BiophotonicsInternational 11:36-42; Rasband, W. S. 1997-2005. ImageJ. NationalInstitutes of Health). Replication in the presence of the expressioncassettes indicated on the left was normalized to amount of replicatedDNA in the presence of the empty expression (set to 100). Cut off valuesof ≧25%, ≧50% and ≧65% indicate strong (black bars), moderate (graybars), and weak interference (white bars), respectively. SomeN-TrxA-peptides show no significant interference (also in white bars).Each assay was performed in triplicate with the error bars correspondingto 2 standard errors.

FIG. 6 shows results of studies designed to study interaction withreplication proteins from a heterologous geminivirus. SelectedN-TrxA-peptides were tested for interaction with CaLCuV AL1. FIG. 6Aprovides a key for the aptamers on the plates in FIG. 6 B. The negativeprey control AD:Jun is marked by a “C”. FIG. 6B provides results ofyeast cells containing the selected aptamers and the TAL1₁₋₁₃₀ (left),TAL1₁₋₃₅₂ (center) and CaAL1₁₋₃₄₉ (right) baits were analyzed for growthon Gal-HWUL medium.

FIG. 7 presents results showing the statistical significance of pairwisealignments. Pairwise alignments were performed for 100 sets of threerandom databases of computer-generated 20-mers containing 88, 31 or 57members. The frequencies of hits were compared to equivalent alignmentsof the databases corresponding to All, Interfering or Non-interferingN-TrxA peptides, respectively. (A) The left panel shows the frequencydistribution (expected mean=54) of a random 20-mer of having at leastone hit against a database comprised of 88 random 20-mers. The rightpanel shows the frequency distribution (expected mean=101) of the totalnumber of hits per peptide for all the 88 random 20-mers. The dashedlines represent the observed values for the All N-TrxA-peptides databasefor each analysis. Similar analyses were performed for the Interferingand Non-interfering TrxA peptide databases and their random 20-mercontrol databases (not shown). In FIG. 7B, the observed and expectedmeans and standard errors of the pairwise alignments of the three TrxApeptide databases are given. The observed values for the three databasesare significantly higher than the expected values derived from therandom 20-mer databases (p values<0.0001).

FIG. 8 presents motifs involved in All binding and replicationinterference. FIG. 8A presents consensus sequences corresponding tomotifs identified in pairwise alignments of the 88 TrxA-peptidesdescribed herein. Bold typeface indicates invariant residues, normaltypeface marks amino acids conserved in a majority of group members, andX represents any amino acid. The number of members and interferingpeptides in each group are listed on the right. (See FIGS. 9 and 10 forsequence alignments). FIG. 8B provides WebLogo representation of Motif24. The amino acid type and position is shown in the X-axis. The overallheight plotted on the Y-axis of the amino acid stacks indicates thesequence conservation at a given position, while the height ofindividual symbols within a stack indicates the relative frequency of anamino acid at that position (Crooks GE, G. Hon, J. M. Chandonia, S. E.Brenner 2004. WebLogo: A sequence logo generator. Genome Res.14:1188-1190; Schneider T. D., and R. M. Stephens 1990. Sequence Logos:A new way to display consensus sequences. Nucleic Acids Res.18:6097-6100). Amino acids are color coded according to their type asbasic (blue), hydrophobic (black), polar/non polar (green) and acidic(red) (Bogan, A. A., and K. S. Thorn 1998. Anatomy of hot spots inprotein interfaces. J. Mol. Biol. 280:1-9; Glaser, F., D. Steinberg, I.Vakser, and N. Ben-Tal 2001. Residue frequencies and pairing preferencesat protein-protein interfaces. Proteins 43:89-102).

FIG. 9 presents sequence alignment of Motif 24 peptides. Peptidescontaining Motif 24 were classified as interfering or non-interfering,and each class was aligned using Vector NTI-AlignX. Coding of amino acidsimilarities from Vector NTI-AlignX is: UPPERCASE normal—non-similarresidues; UPPERCASE bold—a consensus residue derived from a block ofsimilar residues; lowercase bold and shaded—a consensus residue derivedfrom the occurrence of greater than 50% of a single residue; UPPERCASEbold and shaded—a consensus derived from a completely conserved residue;and lowercase bold—a residue weakly similar to a consensus residue at agiven position.

FIG. 10 presents sequence alignments of selected motifs. Motifs 1, 4,20, 25 and 27 include primarily interfering peptides (in bold). Motif 28includes mostly noninterfering peptides (normal). Consensus color codepresented above (FIG. 9).

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Except as otherwise indicated, standard methods can be used for theproduction of viral and non-viral vectors, manipulation of nucleic acidsequences, production of transformed cells, and the like according tothe present invention. Such techniques are known to those skilled in theart. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. Also as used herein, “and/or”refers to and encompasses any and all possible combinations of one ormore of the associated listed items, as well as the lack of combinationswhen interpreted in the alternative (“or”).

As used herein, “aptamers” may be small peptide or nucleic acidmolecules that specifically recognize and bind proteins, and in someinstances, regulate a protein of interest, for example, decreaseactivity of the protein. In particular, peptide aptamers are recombinantproteins that have been selected for specific binding to a targetprotein (Hoppe-Seyler, Crnkovic-Mertens et al. 2004). They generallyinclude a short peptide domain inserted into a supporting proteinscaffold that enhances both specificity and affinity by conformationallyconstraining the peptide sequence (Colas, Cohen et al. 1996; Cohen,Colas et al. 1998; Buerger, Nagel-Wolfrum et al. 2003). Bacterialthioredoxin (Trx), which is rendered inactive by insertion of thepeptide sequence into its active site, is the most commonly usedscaffold because of its small size (12 kD), stability, solubility andknown 3D structure. In some embodiments of the present invention,“aptamer” may be used to designate the peptide in the scaffold proteinwhile “peptide” may refer to the inserted sequence.

“Amino acid sequence” as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or partially or completely synthetic molecules.Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence,” and like terms, are not meant to limit the amino acidsequence to the complete, native amino acid sequence associated with therecited protein molecule.

A “functional fragment” of an amino acid sequence as used herein, refersto a portion of the amino acid sequence that retains at least onebiological activity normally associated with that amino acid sequence.

In particular embodiments, a “functional variant” of an amino acidsequence as used herein, refers to no more than one, two, three, four,five, six, seven, eight, nine or ten amino acid substitutions in thesequence of interest. The functional variant retains at least onebiological activity normally associated with that amino acid sequence.In particular embodiments, the “functional variant” retains at leastabout 40%, 50%, 60%, 75%, 85%, 90%, 95% or more biological activitynormally associated with the full-length amino acid sequence. In otherembodiments, a “functional variant” is an amino acid sequence that is atleast about 60%, 70%, 80%, 90%, 95% 97% or 98% similar to thepolypeptide sequence disclosed herein (or fragments thereof).

“Polypeptide” as used herein, is used interchangeably with “protein,”and refers to a polymer of amino acids (dipeptide or greater) linkedthrough peptide bonds. Thus, the term “polypeptide” includes proteins,oligopeptides, protein fragments, protein analogs and the like. The term“polypeptide” contemplates polypeptides as defined above that areencoded by nucleic acids, are recombinantly produced, are isolated froman appropriate source, or are synthesized.

“Fusion protein” as used herein, refers to a protein produced when twoheterologous nucleotide sequences or fragments thereof coding for two(or more) different polypeptides, or fragments thereof, are fusedtogether in the correct translational reading frame. The two or moredifferent polypeptides, or fragments thereof, include those not foundfused together in nature and/or include naturally occurring mutants.

“Isolated” nucleic acid as used herein, refers to a nucleic acidseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, such as forexample, the cell or viral structural components or other polypeptidesor nucleic acids commonly found associated with the nucleic acid.Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

“Vector” as used herein, refers to a viral or non-viral vector that isused to deliver a nucleic acid to a cell, protoplast, tissue or subject.

“Transgenic” as used herein, refers a plant that comprises a foreignnucleic acid incorporated into the genetic makeup of the plant, such asfor example, by stable integration into the nuclear genome.

“Plant cell” as used herein, refers to plant cells, plant protoplastsand plant tissue cultures, plant calli, plant clumps, and plant cellsthat are intact in plants or parts of plants, such as leaves, pollen,embryos, cotyledon, hypocotyl, roots, root tips, anthers, flowers andparts thereof, ovules, shoots, stems, stalks, pith, capsules, and thelike.

“Resistance to a virus infection” as used herein, refers to the reducedsusceptibility of a plant or animal subject to viral infection ascompared with a control susceptible plant or animal subject underconditions of infestation. “Resistance” can refer to reduced onset,severity, duration and/or spread of viral infection.

In embodiments of the present invention, a polypeptide comprises,consists essentially of or consists of: (a) the amino acid sequence ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86,SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96,SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ IDNO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115,SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ IDNO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,SEQ. ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ IDNO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO: 137, SEQ ID NO:138, SEQID NO:139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO:143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ IDNO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166,SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ IDNO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180,SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ IDNO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, or any combination thereof;(b) a functional fragment of any of the amino acid sequences recitedabove that bind to a viral replication protein (Rep); and (c) afunctional variant of any of the amino acid sequences of (a) or (b) thatbinds to a viral replication protein (Rep).

Moreover, polypeptides of the invention encompass those amino acids thathave at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher aminoacid sequence similarity with the polypeptide sequences specificallydisclosed herein (or fragments thereof). As is known in the art, anumber of different programs can be used to identify whether a nucleicacid or polypeptide has sequence identity or similarity to a knownsequence. Sequence identity and/or similarity can be determined usingstandard techniques known in the art, including, but not limited to, thelocal sequence identity algorithm of Smith & Waterman, Adv. Appl. Math.2, 482 (1981), by the sequence identity alignment algorithm of Needleman& Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similaritymethod of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85,2444 (1988),by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequenceprogram described by Devereux at al., Nucl. Acid Res. 12, 387-395(1984), preferably using the default settings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35, 351-360 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5, 151-153 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularlyuseful BLAST program is the WU-BLAST-2 program which was obtained fromAltschul et al., Methods in Enzymology, 266, 460-480 (1996);http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, which are preferably set to the default values. Theparameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values can be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., (1997) Nucleic Acids Res. 25, 3389-3402. A percentage amino acidsequence identity value can be determined by the number of matchingidentical residues divided by the total number of residues of the“longer” sequence in the aligned region. The “longer” sequence is theone having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment can include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the polypeptides specifically disclosed herein,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

The present invention also encompasses functional fragments of thepolypeptides disclosed herein. A functional fragment of an amino acidsequence recited above retains at least one of the biological activitiesof the unmodified sequence, for example, binding to the Rep proteinand/or inhibiting viral replication. In some embodiments, the functionalfragment of the amino acid sequence retains all of the activitiespossessed by the unmodified sequence. By “retains” biological activity,it is meant that the amino acid sequence retains at least about 10%,20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, ofone or more biological activities of the native amino acid sequence (andcan even have a higher level of activity than the native amino acidsequence). A “non-functional” amino acid sequence is one that exhibitsessentially no detectable biological activity normally associated withthe amino acid sequence (e.g., at most, only an insignificant amount,e.g., less than about 10% or even 5%).

The invention further provides functional variants of the polypeptidesdisclosed herein. In particular embodiments, a functional variant of anamino acid sequence recited above has no more than one, two, three,four, five or six amino acid substitutions in the amino acid sequence ofinterest. In some embodiments, one, more than one, or all of the aminoacid substitutions are conservative substitutions. In other embodiments,amino acid substitutions facilitate binding affinity of the polypeptideto the Rep protein and/or improve inhibitory properties. In otherembodiments, a functional variant has no more than 1, 2, 3, 4, 5 or 6amino acid substitutions, insertions and/or deletions in the amino acidsequence of interest. In general, those skilled in the art willappreciate that minor deletions, insertions or substitutions may be madeto the amino acid sequences of peptides of the present invention withoutunduly adversely affecting the activity thereof. Thus, polypeptidescontaining such deletions or substitutions are a further aspect of thepresent invention. In polypeptides containing substitutions orreplacements of amino acids, one or more amino acids of a polypeptidesequence may be replaced by one or more other amino acids wherein suchreplacement does not affect the function of that sequence. Such changescan be guided by known similarities between amino acids in physicalfeatures such as charge density, hydrophobicity/hydrophilicity, size andconfiguration, so that amino acids are substituted with other aminoacids having essentially the same functional properties. For example:Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu,Met, or Ile, preferably Ala or Leu; Leu may be replaced with Ala, Val orIle, preferably Val or Ile; Gly may be replaced with Pro or Cys,preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met,preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, orMet, preferably Pro or Met; Met may be replaced with Pro or Cys,preferably Cys; His may be replaced with Phe or Gln, preferably Phe; Phemay be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may bereplaced with His, Phe or Trp, preferably Phe or Trp; Trp may bereplaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Glnor Ser, preferably Gln; Gln may be replaced with His, Lys, Glu, Asn, orSer, preferably Asn or Ser; Ser may be replaced with Gln, Thr, Pro, Cysor Ala; Thr may be replaced with Gln or Ser, preferably Ser; Lys may bereplaced with Gln or Arg; Arg may be replaced with Lys, Asp or Glu,preferably Lys or Asp; Asp may be replaced with Lys, Arg, or Glu,preferably Arg or Glu; and Glu may be replaced with Arg or Asp,preferably Asp. Once made, changes can be routinely screened todetermine their effects on function.

In particular embodiments, the functional fragment or variant comprisesone or more of the conserved structural motifs of the polypeptidesspecifically disclosed herein (See FIGS. 7-10).

In some embodiments, the polypeptides bind anywhere in the Rep protein.In some other embodiments, the polypeptide binds to the catalytic domainfor DNA cleavage of the Rep protein. Binding of the polypeptide to theRep protein can occur in one or more DNA cleavage motifs (Motif I, MotifII and Motif III) located in the Rep N-terminus (FIG. 1). In certainembodiments, the polypeptide binds to Motif III within the catalyticdomain for DNA cleavage. In other representative embodiments, thepolypeptide binds to the DNA binding domain of the Rep protein or anyother conserved region of the Rep protein (e.g., the N-terminalportion). In still other embodiments, the polypeptide binds to the Repprotein and further inhibits viral replication.

The polypeptides and fusion proteins can bind to a viral Rep protein andoptionally inhibit replication and/or infection. The viruses can includeany single-stranded eukaryotic DNA virus employing a rolling circlereplication mechanism. In representative embodiments, the virus is aplant pathogen or an animal pathogen. In certain embodiments, the virusis a geminivirus, a nanovirus, or a circovirus. In some embodiments,viral infection can be caused by a combination of viruses, i.e., is amixed infection. In some embodiments, the virus is a tomato goldenmosaic virus (TGMV), a cabbage leaf curl virus (CbLCV) or a combinationthereof.

Infectious clones for a variety of geminiviruses, nanoviruses andcircoviruses are available in the art. See, e.g., Table 1, whichprovides sequences for geminivirus, nanovirus and circovirus typemembers. The nucleic acid sequences of other infectious clones areavailable at http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fr-fst-g.htm. Seealso Hill et al., Virology 250, 283-292 (1998); Kong and Hanley-Bowdoin,Plant Cell 14, 1817-1832 (2002) (CbLCV); Sangare et al., Mol Breeding 5,95-102 (1999) (ACMV and EACMV); Petty et al., Virology 277, 429-438(2000); Hanley-Bowdoin, Plant Cell 1, 1057-1067 (2002) (TGMV); and Kuniket al., BioTechnology 12, 500-504 (1994) (TYLCV).

TABLE 1 Biological information for geminivirus,nanovirus and circovirus type members NCBI Acc. Family GenusType Species Abbrev ICTV virus code Motif 3 No. GeminiviridaeMastrevirus Maize streak virus (MSV) 00.029.0.01.001. VRDYILKEPL Y00514Curtovirus Beet curly top virus (BCTV) 00.029.0.02.001 VKSYVDKDGD X04144Begomovirus Bean golden mosaic virus (BGMV-PR) 00.029.0.03.001VKEYIDKDGV M10070 Topocuvirus Tomato pseudo-curly (PCTV) 00.029.0.04.001VNSYVDKDGD X84735 top virus Circoviridae CircovirusPorcine circovirus type 1 (PCV1) 00.016.0.01.001 NKEYCSKEGH U49186Gyrovirus Chicken anaemia virus (CAV) 00.016.0.02.001 NLTYVSKIGG M55918Nanoviridae Nanovirus Subterranean clover stunt virus (SCSV)00.093.0.01.001. AQLYAMKEDS U16730 Babuvirus Banana bunchy top virus(BBTV) 00.093.0.02.001. ARSYCMKEDT S56276 Motif III sequences of virustype members Rep proteins. Motif III sequences of Rep proteins fromgeminiviruses, nanoviruses and circoviruses are shown. The invariant Yand K residues are in bold type. Geminiviruses are subgrouped asbegomoviruses, curtoviruses, topocuviruses or mastreviruses. See VirusTaxonomy: The Seventh Report of the International Committee on Taxonomyof Viruses M.H. van Regenmortel, C.M. Fauquet, D.H.L. Bishop et al.(eds.)Academic Press,1024 pp. (2000) San Diego, Wien New York.

In still other embodiments, the invention provides a fusion proteincomprising, consisting essentially of or consisting of the polypeptiderecited above. In some embodiments, the polypeptide conformation isconstrained. In certain embodiments, polypeptides wherein conformationis constrained can bind to the target with higher affinity as comparedto polypeptides wherein conformation is random. In some embodiments, thefusion protein comprises thioredoxin (or a fragment thereof). In stillother embodiments, the fusion proteins binds to Rep and/or inhibitsviral replication. In certain embodiments, the fusion protein reducesgeminivirus replication.

Embodiments of the present invention further provide an isolated nucleicacid comprising, consisting essentially of or consisting of a nucleotidesequence encoding the polypeptides and fusion proteins of the invention.In some embodiments, the isolated nucleic acid comprises a nucleotidesequence encoding the polypeptide recited above. The nucleic acid can beDNA, RNA or a chimera thereof, and can further include naturallyoccurring bases and/or analogs and derivatives of naturally occurringbases. Further, the isolated nucleic acid can be double-stranded,single-stranded or a combination thereof.

In other embodiments, the present invention further provides a vectorcomprising the isolated nucleic acid recited above. In particularembodiments, the vector is an expression vector. In other particularembodiments, the vector is compatible with bacterial, yeast, animal(e.g., mammalian, insect) or plant (e.g., monocot, dicot) cells.Exemplary vectors include but are not limited to plasmids (including theTi plasmid from Agrobacteria), virus vectors, bacterial artificialchromosomes, yeast artificial chromosomes, bacteriophage and the like.

Also provided are cells comprising the isolated nucleic acids, vectors,polypeptides and fusion proteins of the invention. The cell can be anycell known in the art including plant cells and protoplasts, animalcells, bacterial cells, yeast cells, and the like. Further, inparticular embodiments, the cell can be a cultured cell or a cell in anintact plant or subject in vivo.

Exemplary Viruses 1. Geminivirus

The geminiviruses are single-stranded plant DNA viruses. They possess acircular, single-stranded (ss) genomic DNA encapsidated in twinned“geminate” icosahedral particles. The encapsidated ssDNAs are replicatedthrough circular double stranded DNA intermediates in the nucleus of thehost cell, presumably by a rolling circle mechanism. Viral DNAreplication, which results in the synthesis of both single and doublestranded viral DNAs in large amounts, involves the expression of only asmall number of viral proteins that are necessary either for thereplication process itself or facilitates replication or viraltranscription. The geminiviruses therefore appear to rely primarily onthe machinery of the host for viral replication and gene expression.

Geminiviruses are subdivided on the basis of host range in eithermonocots or dicots and whether the insect vector is a leaf hopper, treehopper or a whitefly species. Monocot-infecting geminiviruses, themastreviruses, are transmitted by leaf hoppers and their genomecomprises a single ss DNA component about 2.7 kb in size (monopartitegeminivirus). This type of genome, the smallest known infectious DNA, istypified by wheat dwarf virus, which is one of a number from thesubgroup that have been cloned and sequenced. A few mastreviruses infectdicot species as illustrated by bean yellow dwarf virus. Members of thegeminivirus begomovirus genus infect dicot hosts and are transmitted bythe whitefly. Many possess a bipartite genome comprising similarly sizedDNAs (usually termed A and B) as illustrated by African cassava mosaicvirus (ACMV), tomato golden mosaic virus (TGMV) and potato yellow mosaicvirus. For successful infection of plants, both genomic components arerequired. Some begomoviruses possess single component genomes, asillustrated by tomato yellow leaf curl virus (TYLCV). Somesingle-component begomoviruses are associated with satellite DNAs, asillustrated by tomato leaf curl virus (TYLC). The curtoviruses, typifiedby beet curly top virus, occupy a unique intermediary position betweenthe above two genera as they infect dicots but are transmitted by leafhoppers. The fourth geminivirus genus, the topocuviruses, is comprisedof a single virus, tomato pseudo-curly top virus, which has singlecomponent genome and is transmitted by tree hoppers.

The bipartite geminiviruses contain only the viruses that infect dicots.Exemplary is the African Cassava Mosaic Virus (ACMV) and the TomatoGolden Mosaic Virus (TGMV). TGMV, like ACMV, is composed of two circularDNA molecules of the same size, both of which are required forinfectivity. Sequence analysis of the two genome components reveals sixopen reading frames (ORFs); four of the ORFs are encoded by DNA A andtwo by DNA B. On both components, the ORFs diverge from a conserved 230nucleotide intergenic region (common region) and are transcribedbidirectionally from double stranded replicative form DNA. The ORFs arenamed according to genome component and orientation relative to thecommon region (i.e., left versus right). The AL2 gene producttransactivates expression of the TGMV coat protein gene, which is alsosometimes known as “AR1”. Functions have not yet been attributed to someof the ORFs in the geminivirus genomes. However, it is known thatcertain proteins are involved in the replication of viral DNA (REPgenes). See, e.g., Elmer et al., Nucleic Acids Res. 16, 7043 (1988);Hatta and Francki, Virology 92, 428 (1979).

The A genome component contains all viral information necessary for thereplication and encapsidation of viral DNA, while the B componentencodes functions required for movement of the virus through theinfected plant. The DNA A component of these viruses is capable ofautonomous replication in plant cells in the absence of DNA B wheninserted as a greater than full-length copy into the genome of plantcells, or when a copy is electroporated into plant cells. In monopartitegeminivirus genomes, the single genomic component contains all viralinformation necessary for replication, encapsidation, and movement ofthe virus.

The geminivirus A component carries the Rep (also known as C1, AC1 orALI), the AL2 (also known as C2 or TRAP), AL3 (also known as C3, AC3 orREN), and AR1 (also known as V1 or coat protein) sequences. Thegeminivirus B component carries the BR1 (also known as BV1) and BL1(also known as BC1) sequences. Additionally, monopartite geminivirusesencode a protein that is homologous to the Rep protein of bipartiteviruses.

As used herein, geminiviruses encompass viruses of the GenusMastrevirus, Genus Curt virus, Genus Topocuvirus and Genus Begomovirus.Exemplary geminiviruses include, but are not limited to, Abutilon MosaicVirus, Ageratum Yellow Vein Virus, Bhendi Yellow Vein Mosaic virus,Cassava African Mosaic Virus, Chino del Tomato Virus, Cotton LeafCrumple Virus, Croton Yellow Vein Mosaic Virus, Dolichos Yellow MosaicVirus, Horsegram Yellow Mosaic Virus, Jatropha Mosaic virus, Lima BeanGolden Mosaic Virus, Melon Leaf Curl Virus, Mung Bean Yellow MosaicVirus, Okra Leaf Curl Virus, Pepper Hausteco Virus, Potato Yellow MosaicVirus, Rhynchosia Mosaic Virus, Squash Leaf Curl Virus, Tobacco LeafCurl Virus, Tomato Australian Leaf Curl Virus, Tomato Indian Leaf CurlVirus, Tomato Leaf Crumple Virus, Tomato Pseudo-Curly Top Virus, TomatoYellow Leaf Curl Virus, Tomato Yellow Mosaic Virus, Watermelon ChloroticStunt Virus, Watermelon Curly Mottle Virus, Bean Distortion Dwarf Virus,Cowpea Golden Mosaic Virus, Lupin Leaf Curl Virus, Solanum Apical LeafCurling Virus, Soybean Crinkle Leaf Virus, Chloris Striate Mosaic Virus,Digitaria Striate Mosaic Virus, Digitaria Streak Virus, MiscanthusStreak Virus, Panicum Streak Virus, Pasalum Striate Mosaic Virus,Sugarcane Streak Virus, Tobacco Yellow Dwarf Virus, Cassava IndianMosaic Virus, Serrano Golden Mosaic Virus, Tomato Golden Mosaic Virus,Cabbage Leaf Curl Virus, Bean Golden Mosaic Virus, Pepper Texas Virus,Tomato Mottle Virus, Euphorbia Mosaic Virus, African Cassava MosaicVirus, Bean Calico Mosaic Virus, Wheat Dwarf Virus, Cotton Leaf CurlVirus, Maize Streak Virus, and any other virus designated as aGeminivirus by the International Committee on Taxonomy of Viruses(ICTV). In particular embodiments, the geminivirus is a Tomato GoldenMosaic Virus (TGMV), a Cabbage Leaf Curl Virus (CbLCV) or a combinationthereof.

2. Nanovirus

Nanovirus Rep proteins differ from those of members of the Geminivirusesin being smaller (about 33 kDa), having a slightly distinct dNTP-bindingmotif and lacking the Rb-binding motif. Moreover, the Nanoviruses aredistinct from Geminivirus particle morphology, genome size, number andsize of DNA components, and mode of transcription. The Nanoviruses havea conserved nona-nucleotide motif at the apex of the stem-loop sequence,which is consistent with the operation of a rolling circle model for DNAreplication.

As used herein, Nanoviruses include, but are not limited to, BananaBunchy Top Virus (BBTV), Coconut Foliar Decay Virus, Faba Bean NecroticYellows Virus (FBNYN), Milk Vetch Dwarf Virus (MVDV), subterraneanclover stunt virus (SCSV), and Ageratum yellow vein virus (AYVV) and anyother virus designated as a Nanovirus by the International Committee onTaxonomy of Viruses (ICTV).

3. Circovirus

Circoviruses infect animal species and are characterized as round,non-enveloped virions with mean diameters from 17 to 23.5 nm containingcircular ssDNA. The ssDNA genome of the circoviruses represent thesmallest viral DNA replicons known. As disclosed in WO 99/45956, atleast six viruses have been identified as members of the familyaccording to The Sixth Report of the International Committee for theTaxonomy of Viruses (Lukert, et al. (1995) Arch. Virol. 10 Suppl.:166-168).

As used herein, Circoviruses include, but are not limited to, members ofthe Circoviridae family including chicken anemia virus (CAV), beak andfeather disease virus (BFDV), porcine circovirus type 1 (PCV1), porcinecircovirus type 2 (PCV2) and pigeon circovirus and any other virusdesignated as a nanovirus by the ICTV. Embodiments of the presentinvention further provide a transgenic plant or plant cell comprisingthe isolated nucleic acid recited above. The plant or cell can be stablytransformed with the isolated nucleic acid. Additionally, embodiments ofthe invention provide a plurality of plants or cells comprising theisolated nucleic acid recited above. In other representativeembodiments, the isolated nucleic acid is flanked by a T-DNA bordersequence, optionally by 5′ and 3′ T-DNA border sequences. In someembodiments, the invention provides a plant cell or plant comprising thepolypeptides or fusion proteins of the present invention. In otherembodiments, the invention provides a plurality of plant Cells or plantscomprising the polypeptides or fusion proteins of the present invention.

Plants can be transformed according to the present invention using anysuitable method known in the art. Intact plants, plant tissue, explants,meristematic tissue, protoplasts, callus tissue, cultured cells, and thelike may be used for transformation depending on the plant species andthe method employed. In a preferred embodiment, intact plants areinoculated using microprojectiles carrying a nucleic acid to betransferred into the plant. The site of inoculation will be apparent toone skilled in the art; leaf tissue is one example of a suitable site ofinoculation. In some embodiments, intact plant tissues or plants areinoculated, without the need for regeneration of plants (e.g., fromcallus).

Exemplary transformation methods include biological methods usingviruses and Agrobacterium, physicochemical methods such aselectroporation, polyethylene glycol, ballistic bombardment,microinjection, floral dip method and the like.

In one form of direct transformation, the vector is microinjecteddirectly into plant cells by use of micropipettes to mechanicallytransfer the recombinant DNA (Crossway, Mol. Gen. Genetics 202: 179(1985)).

In another protocol, the genetic material is transferred into the plantcell using polyethylene glycol (Krens, et al. Nature 296, 72 (1982)).

In still another method, protoplasts are fused with minicells, cells,lysosomes, or other fusible lipid-surfaced bodies that contain thenucleotide sequence to be transferred to the plant (Fraley, et al.,Proc. Natl. Acad. Sci. USA 79, 1859 (1982)).

DNA may also be introduced into the plant cells by electroporation(Fromm et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)). In thistechnique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide and regenerate. One advantage of electroporationis that large pieces of DNA, including artificial chromosomes, can betransformed by this method.

Viral vectors include RNA and DNA viral vectors (e.g., geminivirus,badnavirus, nanoviruses and caulimovirus vectors).

Ballistic transformation typically comprises the steps of: (a) providinga plant tissue as a target; (b) propelling a microprojectile carryingthe heterologous nucleotide sequence at the plant tissue at a velocitysufficient to pierce the walls of the cells within the tissue and todeposit the nucleotide sequence within a cell of the tissue to therebyprovide a transformed tissue. In some particular embodiments of theinvention, the method further includes the step of culturing thetransformed tissue with a selection agent. In particular embodiments,the selection step is followed by the step of regenerating transformedplants from the transformed tissue. As noted below, the technique may becarried out with the nucleotide sequence as a precipitate (wet orfreeze-dried) alone, in place of the aqueous solution containing thenucleotide sequence.

Any ballistic cell transformation apparatus can be used in practicingthe present invention. Exemplary apparatus are disclosed by Sandford etal. (Particulate Science and Technology 5, 27 (1988)), Klein et al.(Nature 327, 70 (1987)), and in EP 0 270 356. Such apparatus have beenused to transform maize cells (Klein et al., Proc. Natl. Acad. Sci. USA85, 4305 (1988)), soybean callus (Christou et al., Plant Physiol. 87,671 (1988)), McCabe et al., BioTechnology 6, 923 (1988), yeastmitochondria (Johnston et al., Science 240, 1538 (1988)), andChlamydomonas chloroplasts (Boynton et al., Science 240, 1534 (1988)).

Alternatively, an apparatus configured as described by Klein et al.(Nature 70, 327 (1987)) may be utilized. This apparatus comprises abombardment chamber, which is divided into two separate compartments byan adjustable-height stopping plate. An acceleration tube is mounted ontop of the bombardment chamber. A macroprojectile is propelled down theacceleration tube at the stopping plate by a gunpowder charge. Thestopping plate has a borehole formed therein, which is smaller indiameter than the microprojectile. The macroprojectile carries themicroprojectile(s), and the macroprojectile is aimed and fired at theborehole. When the macroprojectile is stopped by the stopping plate, themicroprojectile(s) is propelled through the borehole. The target tissueis positioned in the bombardment chamber so that a microprojectile(s)propelled through the bore hole penetrates the cell walls of the cellsin the target tissue and deposit the nucleotide sequence of interestcarried thereon in the cells of the target tissue. The bombardmentchamber is partially evacuated prior to use to prevent atmospheric dragfrom unduly slowing the microprojectiles. The chamber is only partiallyevacuated so that the target tissue is not desiccated duringbombardment. A vacuum of typically between about 400 to about 800millimeters of mercury is suitable.

In alternative embodiments, ballistic transformation is achieved withoutuse of microprojectiles. For example, an aqueous solution containing thenucleotide sequence of interest as a precipitate may be carried by themacroprojectile (e.g., by placing the aqueous solution directly on theplate-contact end of the macroprojectile without a microprojectile,where it is held by surface tension), and the solution alone propelledat the plant tissue target (e.g., by propelling the macroprojectile downthe acceleration tube in the same manner as described above). Otherapproaches include placing the nucleic acid precipitate itself (“wet”precipitate) or a freeze-dried nucleotide precipitate directly on theplate-contact end of the macroprojectile without a microprojectile. Inthe absence of a microprojectile, it is believed that the nucleotidesequence must either be propelled at the tissue target at a greatervelocity than that needed if carried by a microprojectile, or thenucleotide sequenced caused to travel a shorter distance to the targettissue (or both).

The nucleotide sequence can be carried on a microprojectile. Themicroprojectile may be formed from any material having sufficientdensity and cohesiveness to be propelled through the cell wall, giventhe particle's velocity and the distance the particle must travel.Non-limiting examples of materials for making microprojectiles includemetal, glass, silica, ice, polyethylene, polypropylene, polycarbonate,and carbon compounds (e.g., graphite, diamond). Metallic particles arecurrently preferred. Non-limiting examples of suitable metals includetungsten, gold, and iridium. The particles should be of a sizesufficiently small to avoid excessive disruption of the cells theycontact in the target tissue, and sufficiently large to provide theinertia required to penetrate to the cell of interest in the targettissue. Particles ranging in diameter from about one-half micrometer toabout three micrometers are suitable. Particles need not be spherical,as surface irregularities on the particles may enhance their DNAcarrying capacity.

The nucleotide sequence may be immobilized on the particle byprecipitation. The precise precipitation parameters employed will varydepending upon factors such as the particle acceleration procedureemployed, as is known in the art. The carrier particles may optionallybe coated with an encapsulating agents such as polylysine to improve thestability of nucleotide sequences immobilized thereon, as discussed inEP 0 270 356 (column 8).

Alternatively, plants may be transformed using Agrobacterium tumefaciensor Agrobacterium rhizogenes, preferably Agrobacterium tumefaciens.Agrobacterium-mediated gene transfer exploits the natural ability of A.tumefaciens and A. rhizogenes to transfer DNA into plant chromosomes.Agrobacterium is a plant pathogen that transfers a set of genes encodedin a region called T-DNA of the Ti and Ri plasmids of A. tumefaciens andA. rhizogenes, respectively, into plant cells. The typical result oftransfer of the Ti plasmid is a tumorous growth called a crown gall inwhich the T-DNA is stably integrated into a host chromosome. Integrationof the Ri plasmid into the host chromosomal DNA results in a conditionknown as “hairy root disease”. The ability to cause disease in the hostplant can be avoided by deletion of the genes in the T-DNA without lossof DNA transfer and integration. The DNA to be transferred is attachedto border sequences that define the end points of an integrated T-DNA.

Gene transfer by means of engineered Agrobacterium strains has becomeroutine for many dicotyledonous plants. Some difficulty has beenexperienced, however, in using Agrobacterium to transformmonocotyledonous plants, in particular, cereal plants. However,Agrobacterium mediated transformation has been achieved in severalmonocot species, including cereal species such as rye (de la Pena etal., Nature 325, 274 (1987)), maize (Rhodes et al., Science 240, 204(1988)), and rice (Shimamoto et al., Nature 338, 274 (1989)).

While the following discussion will focus on using A. tumefaciens toachieve gene transfer in plants, those skilled in the art willappreciate that this discussion also applies to A. rhizogenes.Transformation using A. rhizogenes has developed analogously to that ofA. tumefaciens and has been successfully utilized to transform, forexample, alfalfa, Solanum nigrum L., and poplar. U.S. Pat. No. 5,777,200to Ryals et al. As described by U.S. Pat. No. 5,773,693 to Burgess etal., it is preferable to use a disarmed A. tumefaciens strain (asdescribed below), however, the wild-type A. rhizogenes may be employed.An illustrative strain of A. rhizogenes is strain 15834.

The Agrobacterium strain is typically modified to contain the nucleotidesequences to be transferred to the plant. The nucleotide sequence to betransferred is incorporated into the T-region and is typically flankedby at least one T-DNA border sequence, preferably two T-DNA bordersequences. A variety of Agrobacterium strains are known in the art, andcan be used in the methods of the invention. See, e.g., Hooykaas, PlantMol. Biol. 13, 327 (1989); Smith et al., Crop Science 35, 301 (1995);Chilton, Proc. Natl. Acad. Sci. USA 90, 3119 (1993); Mollony et al.,Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al., NatureBiotechnol. 14, 745 (1996); and Komari et al., The Plant Journal 10, 165(1996).

In addition to the T-region, the Ti (or Ri) plasmid contains a virregion. The vir region is important for efficient transformation, andappears to be species-specific.

Two exemplary classes of recombinant Ti and Ri plasmid vector systemsare commonly used in the art. In one class, called “cointegrate,” theshuttle vector containing the gene of interest is inserted by geneticrecombination into a non-oncogenic Ti plasmid that contains both thecis-acting and trans-acting elements required for plant transformationas, for example, in the PMLJ1 shuttle vector of DeBlock et al., EMBO J.3, 1681 (1984), and the non-oncogenic Ti plasmid pGV2850 described byZambryski et al., EMBO J. 2, 2143 (1983). In the second class or“binary” system, the gene of interest is inserted into a shuttle vectorcontaining the cis-acting elements required for plant transformation.The other necessary functions are provided in trans by the non-oncogenicTi plasmid as exemplified by the pBIN19 shuttle vector described byBevan, Nucleic Acids Research 12, 8711 (1984), and the non-oncogenic Tiplasmid PAL4404 described by Hoekma, et al., Nature 303, 179 (1983).

Binary vector systems have been developed where the manipulated disarmedT-DNA carrying the heterologous nucleotide sequence of interest and thevir functions are present on separate plasmids. In this manner, amodified T-DNA region comprising foreign DNA (the nucleic acid to betransferred) is constructed in a small plasmid that replicates in E.coli. This plasmid is transferred conjugatively in a tri-parental matingor via electroporation into A. tumefaciens that contains a compatibleplasmid with virulence gene sequences. The vir functions are supplied intrans to transfer the T-DNA into the plant genome. Such binary vectorsare useful in the practice of the present invention.

Plant cells may be transformed with Agrobacteria by any means known inthe art, e.g., by co-cultivation with cultured isolated protoplasts, ortransformation of intact cells or tissues. The first generally utilizesan established culture system that allows for culturing protoplasts andsubsequent plant regeneration from cultured protoplasts. Identificationof transformed cells or plants is generally accomplished by including aselectable marker in the transforming vector, or by obtaining evidenceof successful bacterial infection.

In plants stably transformed by Agrobacteria-mediated transformation,the nucleotide sequence of interest is incorporated into the plantgenome, typically flanked by at least one T-DNA border sequence. In someembodiments, the nucleotide sequence of interest is flanked by two T-DNAborder sequences.

Alternatively, transgenic plants may be produced using thewell-established ‘floral dip’ method (See, e.g., Clough and Bent (1998)Plant Journal 16:735). In one representative protocol, plants are grownin soil until the primary inflorescence is about 10 cm tall. The primaryinflorescence is cut to induce the emergence of multiple secondaryinflorescences. The inflorescences of these plants are dipped in asuspension of Agrobacterium containing the vector of interest. After thedipping process, the plants are grown to maturity and the seeds areharvested. Transgenic seeds from these treated plants are selected bygermination in soil under selective pressure (e.g., using the chemicalbialaphos). Transgenic plants containing the selectable marker survivetreatment and are transplanted to individual pots for subsequentanalysis. See Bechtold, N. and Pelletier, G. Methods Mol Biol 82,259-266 (1998); Chung, M. H. et al. Transgenic Res 9, 471-476 (2000);Clough, S. J. and Bent, A. F. Plant J 16, 735-743 (1998); Mysore, K. S.et al. Plant J 21, 9-16 (2000); Tague, B. W. Transgenic Res 10, 259-267(2001); Wang, W. C. et al. Plant Cell Rep 22, 274-281 (2003); Ye, G. N.et al. Plant J., 19:249-257 (1999).

Plant cells, which have been transformed by any method known in the art,can also be regenerated to produce intact plants using known techniques.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMilan Publishing Co.New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II,1986). It is known that practically all plants can be regenerated fromcultured cells or tissues, including but not limited to, all majorspecies of sugar-cane, sugar beet, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently root.Alternatively, somatic embryo formation can be induced in the callustissue. These somatic embryos germinate as natural embryos to formplants. The culture media will generally contain various amino acids andplant hormones, such as auxin and cytokinins. It is also advantageous toadd glutamic acid and proline to the medium, especially for such speciesas corn and alfalfa. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these threevariables are controlled, then regeneration is usually reproducible andrepeatable.

A large number of plants have been shown capable of regeneration fromtransformed individual cells to obtain transgenic whole plants.

The regenerated plants are transferred to standard soil conditions andcultivated in a conventional manner. The plants are grown and harvestedusing conventional procedures.

The particular conditions for transformation, selection and regenerationmay be optimized by those of skill in the art. Factors that affect theefficiency of transformation include the species of plant, the tissueinfected, composition of the media for tissue culture, selectable markergenes, the length of any of the above-described step, kinds of vectors,and light/dark conditions. Therefore, these and other factors may bevaried to determine what is an optimal transformation protocol for anyparticular plant species. It is recognized that not every species willreact in the same manner to the transformation conditions and mayrequire a slightly different modification of the protocols disclosedherein. However, by altering each of the variables, an optimum protocolcan be derived for any plant species.

The foregoing methods for transformation may be used for producingtransgenic inbred or doubled-haploid lines. Transgenicinbred/doubled-haploid lines could then be crossed, with another(non-transformed or transformed) inbred or doubled-haploid line, inorder to produce a transgenic hybrid plant. Alternatively, a genetictrait which has been engineered into a particular line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts.

Plants that may be employed in practicing the present invention includeany plant (angiosperm or gymnosperm; monocot or dicot).

Exemplary plants include, but are not limited to corn (Zea mays), canola(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice(Oryza sativa), rape (Brassica napus), rye (Secale cereale), sorghum(Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), apple (Malus pumila),blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape(Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach(Prunus persica), plum (Prunus domestica), pear (Pyrus communis),watermelon (Citrullus vulgaris). duckweed (Lemna), oats, barley,vegetables, ornamentals, conifers, and turfgrasses (e.g., forornamental, recreational or forage purposes).

Vegetables include Solanaceous species (e.g., tomatoes; Lycopersiconesculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota),cauliflower (Brassica oleracea), celery (apium graveolens), eggplant(Solanum melongena), asparagus (Asparagus officinalis), ochra(Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), members of the genus.Cucurbita such as Hubbard squash (C. Hubbard), Butternut squash (C.moschata), Zucchini (C. pepo), Crookneck squash (C. crookneck), C.argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis,C. foetidissima, C. lundelliana, and C. martinezii, and members of thegenus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C.cantalupensis), and musk melon (C. melo).

Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophyllahydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips(Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima),and chrysanthemum.

Conifers, which may be employed in practicing the present invention,include, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis).

Turfgrass include, but are not limited to, zoysiagrasses, bentgrasses,fescue grasses, bluegrasses, St. Augustinegrasses, bermudagrasses,bufallograsses, ryegrasses, and orchardgrasses.

Also included are plants that serve primarily as laboratory models,e.g., Arabidopsis.

In some embodiments, the transgenic plant can be a tobacco plant, apotato plant, a soybean plant, a peanut plant, a tomato plant, a melonplant, a cassava plant, a bean plant, a squash plant, a maize plant, acotton plant or a vegetable plant. In certain embodiments, the plant isa cassava plant. In still other embodiments, the present inventionprovides methods of providing resistance against a plant virusinfection, in an agricultural field, comprising planting the field witha crop of plants as recited above. Accordingly, the present inventionprovides a plurality of transgenic plants, such as a crop, having aresistance against a plant virus infection and fields of grasses havingthe same.

Embodiments of the present invention further provide transgenic plantshaving increased resistance to a virus infection from viruses such as ageminivirus, a nanovirus and combinations thereof as compared to anon-transgenic control. Resistance may be evaluated by any suitablemethod known in the art, e.g., measuring inhibition of viralreplication, detecting specific mutations within the genome of the viralagent, detecting and quantifying viral load and measuring surrogatemarkers of viral replication. The term “resistant/resistance” is notintended to indicate that the subject is absolutely immune from viralinfection. Those skilled in the art will appreciate that the degree ofresistance may be assessed with respect to a population of subjects oran entire field of plants. A subject may be considered “resistant” toviral infection if the overall incidence of infection is reduced, evenif particular subjects may be susceptible to disease.

In some embodiments of the present invention, methods of makingtransgenic plants having increased resistance to a virus compriseintroducing an isolated nucleic acid recited above into a plant cell toproduce a transgenic plant, wherein expression of the isolated nucleicacid to produce the polypeptide increases resistance of the transgenicplant to infection by a virus. In particular embodiments, the presentinvention provides methods of making transgenic plants having increasedresistance to a virus, wherein the method comprises providing a plantcell capable of regeneration; transforming the plant cell with anisolated nucleic acid comprising an isolated nucleic acid recited above;and regenerating a transgenic plant from that transformed plant cell,wherein expression of the isolated nucleic acid to produce thepolypeptide increases resistance of the transgenic plant to infection bya virus. In some embodiments, the plant cell can be a tobacco plantcell, a potato plant cell, a soybean plant cell, a peanut plant cell, atomato plant cell, a melon plant cell, a cassava plant cell, a beanplant cell, a squash plant cell, a maize plant cell, a cotton plant cellor a vegetable plant cell. In still other embodiments, the plant cell isstably transformed with the isolated nucleic acid. In certainembodiments, the plant cell is transformed by an Agrobacterium-mediatedtransformation method. In other embodiments, the plant cell istransformed by a biolistic transformation method.

In still other embodiments, the present invention provides methods ofinhibiting viral replication in a plant cell (e.g., a cultured plantcell or protoplast or a plant cell in vivo) comprising introducing anisolated nucleic acid recited above into the plant cell in an amounteffective to inhibit virus replication as compared to non-transgeniccontrol. In some embodiments, virus replication is inhibited by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%,or more. In certain embodiments, the virus can be a geminivirus, ananovirus and combinations thereof.

In still other embodiments, the invention provides methods of inhibitingviral replication in a plant cell by introducing a polypeptide or fusionprotein of the invention into a plant cell. In still other embodiments,the invention provides a method of providing increased resistance to aviral infection comprising introducing a polypeptide or fusion proteinof the invention into a plant.

A further embodiment of the present invention is a method of detecting aviral infection. The method can involve contacting a sample with atleast one polypeptide of the present invention or a fusion proteinthereof and detecting the presence or absence of binding between thepolypeptide and a target, wherein the binding of the polypeptide to thetarget in the sample indicates the presence of a virus.

A sample is intended to include biological or environmental material,which may be suspected of containing a virus of the familyGeminiviridae, Nanoviridae or Circoviridae. A sample suspected ofcontaining a virus is one that may have come in contact with a virus ormay be at risk of having or acquiring a virus. When the virus beingdetected is of the family Circoviridae (or other animal virus asdescribed above), a sample may be blood, plasma, serum, cell products,cell line cultures, cell extracts, cerebrospinal fluid (CSF), tissuehomogenates, urine, organs for transplantation, or semen isolatedpreferably from a bird, such as chicken or pigeon, or a pig. When thevirus being detected is of the family Geminiviridae or Nanoviridae (orother plant viruses as described above), the sample may be a planttissue culture, fruit, leaf, root, stem, or seed. A sample ofenvironmental origin may include, but not be limited to, soil, water,and food samples including canned goods, meats, and animal fodder. It iscontemplated that the method of the invention may be useful in detectingviral contaminants in an environmental sample, viral presence in anorgan being used in a transplantation, or viral infection of plant seedsor tissue cultures.

Upon, binding of a polypeptide with its viral target, excess polypeptideand/or targets that do not bind said polypeptide may be removed bywashing the sample so that bound polypeptide-target complexes areisolated. Subsequently, the presence or absence of binding (i.e., thepresence or absence of polypeptide-target complexes) is measured ordetected. To facilitate the step of detecting the presence of absence ofbinding, the polypeptide of the present invention can be labeled,preferably with a fluorescent or bioluminescent tag. Fluorochromes suchas Phycocyanine, Allophycocyanine, Tricolor, AMCA, Eosin, Erythrosin,Fluorescein, Fluorescein Isothiocyanate Hydroxycoumarin, Rhodamine,Texas Red, Lucifer Yellow, and the like may be attached directly to apolypeptide of the invention through standard groups such as sulfhydrylor primary amine groups. Methods of imaging and analyzing any of theabove-mentioned labels are well-known in the art and the method employedwill vary with the type of analysis being conducted, i.e. individualsamples or multiple sample analyses in high-throughput screens.Measurement of the label can be accomplished using flow cytometry, laserconfocal microscopy, spectrofluorometer, fluorescence microscopy,fluorescence scanners and the like.

Further, a polypeptide of the present invention may be biotinylated anddetection of a biotinylated polypeptide may be performed using any ofthe well-known avidin or streptavidin reagents. Detection ofbiotin-avidin or biotin-streptavidin complexes typically involvesconjugated forms of avidin or streptavidin including, but are notlimited to, enzyme-conjugates (e.g., alkaline phosphatase,β-galactosidase, glucose oxidase, horseradish peroxidase) orfluorescent-conjugates (e.g., 7-amino-4-methylcoumarin-3-acetic (AMCA),fluorescein, phycoerythrin, rhodamine, TEXAS RED®, OREGON GREEN®) orantibodies which specifically bind to avidin or streptavidin.

Antibodies which specifically interact with a polypeptide of the presentinvention can also be used in the detection of binding between saidpolypeptide and its target. As will be understood by one of skill in theart, a bound polypeptide-target complex is contacted with an antibodyspecific for said polypeptide and standard methods for detectingantibodies are employed for detecting binding of the antibody to thepolypeptide-target complex, e.g., spectrofluorometer, fluorescencemicroscopy, immunocytochemistry, western blotting, ELISA, fluorescencescanners, and the like. Other methods for detecting antibodies arewell-known to those of skill in the art (see, e.g., “Methods inImmunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons,1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc.,1964; and Oellerich, M. (1984) J. Clin. Chem. Clin. Biochem.22:895-904).

Subsequently, the presence or absence of a bound polypeptide-targetcomplex is then correlated with the presence or absence of a virus fromwhich the target was derived.

As will be appreciated by one of skill in the art, the detection methodof the invention may be used to detect one or more specific viruses,genera, or family of viruses depending on the specificity of thepolypeptide being used.

In still other embodiments, the products of the present invention can beused for the preparation of a medicament, veterinary or agriculturalproduct.

The present invention is applicable to animal, avian and plant subjects,where appropriate, for medicinal, diagnostic, drug screening,veterinary, or agricultural purposes. For example, geminiviruses andnanoviruses affect plants, circoviruses affect livestock and poultry,and a human circovirus has been identified in patients with Hepatitis C.Animal subjects include, but are not limited to, humans, primates,canines, felines, bovines, caprines, equines, ovines, porcines, rodents(e.g. rats and mice), lagomorphs, and the like, and mammals in utero.Avian subjects include, but are not limited to, chickens, ducks,turkeys, geese, quail, pheasant, ratites (e.g., ostrich) anddomesticated birds (e.g., parrots and canaries), and birds in ovo. Plantsubjects are described above.

Methods of the present invention can be carried out in a manner suitablefor administration or application to the suitable subject.Administration to a plant or a plant cell is described above.Additionally, an isolated nucleic acid vector, polypeptide or fusionprotein of the present invention can be used to formulate pharmaceuticalcompositions comprising a vector of the invention in a pharmaceuticallyacceptable carrier and/or other medicinal agents, pharmaceutical agents,carriers, adjuvants, diluents, etc. For injection, the carrier willtypically be a liquid. For other methods of administration, the carriermay be either solid or liquid. For inhalation administration, thecarrier will be respirable, and will preferably be in solid or liquidparticulate form. As an injection medium, it is preferred to use waterthat contains the additives usual for injection solutions, such asstabilizing agents, salts or saline, and/or buffers.

In general, a “physiologically acceptable carrier” is one that is nottoxic or unduly detrimental to cells. Exemplary physiologicallyacceptable carriers include sterile, pyrogen-free water and sterile,pyrogen-free, phosphate buffered saline. physiologically acceptablecarriers include pharmaceutically acceptable carriers.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject without causing any undesirable biologicaleffects. Thus, such a pharmaceutical composition may be used, forexample, in transfection of a cell ex vivo or in administering a viralparticle or cell directly to a subject.

As used herein, the term “effective amount” refers to an amount of acompound or composition that is sufficient to produce the desiredeffect, which can be a therapeutic or agricultural effect, i.e., aneffect on plant and plant matter as described herein. The effectiveamount will vary with the application for which the compound orcomposition is being employed, the subject, the age and physicalcondition of the subject, the severity of the condition, the duration ofthe treatment, the nature of any concurrent treatment, thepharmaceutically or agriculturally acceptable carrier used, and likefactors within the knowledge and expertise of those skilled in the art.An appropriate “effective amount” in any individual case can bedetermined by one of ordinary skill in the art by reference to thepertinent texts and literature and/or by using routine experimentation.(See, for example for pharmaceutical applications, Remington, TheScience And Practice of Pharmacy (20th Ed. 2000).

Dosages will depend upon the mode of administration, the disease orcondition to be treated, the individual subject's condition, and can bedetermined in a routine manner. See e.g., Remington, The Science AndPractice of Pharmacy (20th Ed. 2000).

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed.

Exemplary modes of administration include oral, rectal, transmucosal,topical, transdermal, in utero (or in ovo), inhalation, parenteral(e.g., intravenous, subcutaneous, intradermal, intramuscular, andintraarticular) administration, and the like, as well as direct tissueor organ injection, alternatively, intrathecal, direct intramuscular,intraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Alternatively, administration may be by local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

As used herein, the term “treat” refers to an action resulting in areduction in the severity of the subject's condition or at least thecondition is partially improved or ameliorated and/or that somealleviation, mitigation or decrease in at least one clinical symptom (oragricultural index for plants) is achieved and/or there is a delay inthe progression of the condition and/or prevention or delay of the onsetof the condition. Thus, the term “treat” refers to both prophylactic andtherapeutic treatment regimes.

As used herein, the term “agriculturally acceptable carrier” refers toadjuvants, e.g., inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in agricultural formulationtechnology.

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

Example 1 Viruses

Geminivirus Rep proteins have been studied extensively, and many of theassays for studying this protein are well-established in the art.Consequently, geminiviruses were used to test the capacity of aptamersto interfere with ssDNA virus replication and infection in eukaryotes.The tomato golden mosaic virus (TGMV) and cabbage leaf curl virus(CbLCV) were primarily studied. TGMV is a bipartite geminivirus thatinfects solanaceous species and encodes a typical Rep protein. CbLCV hasa bipartite genome and is a severe pathogen in brassica. CbLCV isrepresentative of a small group of dicot-infecting geminiviruses thatencode an atypical Rep protein. Other viruses such as ACMV, BCTV, EACMV,MSV, ToMoV and TYLCV may also be useful in the analysis of selectedaptamers. For example, TYLCV is a monopartite geminivirus that causessignificant losses in tomato crops through out the world. Genomic clonesas well as replication and infectivity assays are well established forthese viruses. Together, these viruses can be used to establish theefficacy and breadth of the aptamer resistance strategy for ssDNAviruses.

Example 2 Materials and Methods A. Yeast Plasmids.

The bait and prey plasmids used in this study are listed in Table 2.pNSB1118, the bait plasmid for full-length TGMV AL1 (TAL1₁₋₃₅₂), wasgenerated by cloning a 1.2-Kb fragment with NdeI (trimmed) and BamHIends from pNSB736 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122)into pEG202 (Golemis and Brent (1992) Mol. Cell. Biol. 12:3006-3014)with BamHI and EcoRI (repaired) ends. The same fragment was also ligatedinto pHybLex/Zeo (Invitrogen) digested with PvuII and BamHI to createpNSB1089. The TAL1₁₋₃₅₂ coding sequence from pNSB1089 was introducedinto pYESTrp2 (Invitrogen) as a SacI/XhoI fragment to give the preyplasmid pNSB970. The bait plasmid for truncated TAL1₁₋₁₃₀ (pNSB1162) wasgenerated in two steps. First, an 895-bp EcoRI/BamHI fragment frompNSB603 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122) was ligatedinto the same sites of pNSB1118 to create pNSB1153. Then, pNSB1153 wasdigested with NotI, repaired with E. coli DNA polymerase (Klenowfragment) and religated to delete an 861-bp sequence encoding the TAL1C-terminus. The bait plasmid (pNSB1122) for full-length CaLCuV ALA(CaAL1₁₋₃₄₉) was built by cloning a 1.2-Kb BamHI/XhoI fragment frompNSB909 (Kong and Hanley-Bowdoin (2002) Plant Cell 14:1817-1832) intothe same sites of pEG202.

The β-glucuronidase coding sequence (GUS) from pMON10018 (Monsanto) wascloned into pEG202 as a 2.2-Kb BglII-NotI fragment to give the controlbait plasmid pNSB1120. An EcoRI/NotI fragment encoding TrxA-GST frompNSB1166 (described below) was cloned into the same sites of pYESTrp2 togenerate pNSB1172, a negative control prey plasmid.

TABLE 2 Yeast dihybrid plasmids Insert Cloning vector Yeastselection^(a) Plasmid Bait (DBD) TAL1₁₋₃₅₂ pEG202 (−) Histidine pNSB1118TAL1₁₋₁₃₀ pEG202 (−) Histidine pNSB1162 CaAL1₁₋₃₄₉ pEG202 (−) HistidinepNSB1122 GUS pEG202 (−) Histidine pNSB1120 Prey (AD) TAL1₁₋₃₅₂ pYESTrp2(−) Tryptophan pNSB970 Jun pYESTrp2 (−) Tryptophan pYESTrp-Jun TrxA-GSTpYESTrp2 (−) Tryptophan pNSB1172 ^(a)(−) Histidine, medium lackinghistidine; (−) Tryptophan, medium lacking tryptophan.

B. Plant Expression Plasmids

TrxA-peptide prey plasmids isolated in the TAL1₁₋₃₅₂ screen weredigested with EcoRI-XbaI, and the resulting 412-bp fragments were gelpurified and cloned into pMON921 (Fontes et al. (1994) J. Biol. Chem.269:8459-8465). To eliminate the gel purification step during thecloning of aptamers derived from the AL1₁₋₁₃₀ screen, a polylinker wasinserted into pMON921 and the β-lactamase gene was replaced by theaminoglycoside 3′-phosphotransferase (aphA) coding sequence, whichconfers kanamycin resistance. The polylinker was generated by ligatingthe annealed oligonucleotides LLp27 and LLp28 (Table 3) into pMON921digested with BglII/BamH1 to create pNSB1208. A fragment carrying theaphA gene was amplified from pFGC5941 (Kerschen et al. (2004) FEBS Lett.566:223-228) using the primers LLp29 and LLp30 (Table 3), digested withSmaI/AatII and cloned into pNSB1208 cut with DraI/AatII to generatepNSB1226. The N-TrxA peptides were cloned into pNSB1226 as EcoRI/BamHIfragments. N-TrxA aptamers with internal EcoRI or BamHI sites (N-3,N-71, N-99, N-123, N-149 and N-153) were cloned into pNSB1226 asPCR-generated EcoRI/SacI or Sad inserts using primers LLp41 and LLp42(Table 3).

The TrxA-GST control was generated in two steps. First, the RsrII sitein the active site of the thioredoxin coding sequence (TrxA) wasreconstituted. Two fragments were generated using pJM-1 library DNA astemplate in PCR reactions with primer pairs LLp9/LLp16 and LLp15/LLp12(Table 3.) The PCR products were digested with RsrII and ligated invitro. The resulting 414-bp fragment was digested with EcoRI/BamHI andcloned into the same sites of pBSKSII(−) to give pNSB1151. Bothconstructs were sequenced to verify the integrity of the TrxA sequence.Oligonucleotides LLp55 and LLp56 (Table 3), carrying a 60-bp sequence ofthe glutathione S-transferase gene (GST), were annealed and ligated intoRsrII site of pNSB1151, creating pNSB1166. An EcoRI/BamHI fragment frompNSB1166 was cloned into the same sites of pMON921 to generate pNSB1168.The expression cassette (pNSB866) corresponding to FQ118, a TAL1transdominant negative mutant, has been described before (Orozco et al.(2000) J. Biol. Chem. 275:6114-6122).

TABLE 3 Oligonucleotides Oligonucleotide Target Sequence^(a) ApplicationLLp1 5′-AL1 TGMV A GATGTTTGGCAACCTCCTCTAG Replication LLp2 3′-CP TGMV AGGTCGTTCTTTACCGTTGCAGTAC Replication LLp9 5′-pJM-1TCAATGAGCTCGGTCCTACCCTTATGATGTG Cloning LLp10 5′-pJM-1TTCACCTGACTGACGACAGT Sequencing LLp12 3′-pJM-1ATGGATCCAGGCCTCTGGCGAAGAAGTCC Cloning LLp13 5′-pMON921TCATTTCATTTGGAGAGGACACGC Sequencing LLp14 3′-pMON921CCAATGCCATAATACTCGAACTCA Sequencing LLp15 TrxA-2TACAGCGGTCCGTGCAAAATGATCGCC Cloning LLp16 TrxA-1 CGGACCGCACCACTCTGCCCAGCloning LLp27 pMON921 GATCTGAATTCGCGATCTAGAGAGCTCG Cloning LLp28 pMON921GATCCGAGCTCTCTAGATCGCGAATTCA Cloning LLp29 5′-aphAAATTCGGACGTCGCTCCGTCGATACTATGTTATACGCC Cloning LLp30 3′-aphAATGACCCGGGGACGCTCAGTGGAACGAAAACTCACG Cloning LLp39 5′-Npt IIGGCGATAGAAGGCGATGCGCTGCG Replication LLp40 3′-Npt IITGCACGCAGGTTCTCCGGCCGCT Replication LLp41 5′-pJM-1AAGAGCTCAGTACTCCTACCCTTATGATGTGCCA Cloning LLp42 3′-pJM-1TTGAGCTCCTCTGGCGAAGAAGTCCA Cloning LLp55 5′-GST 20merGTCCGGAGCTCCCTATACTAGGTTATTGGAAAATTAAGGGC Cloning CTTGTGCAACCCACTCGCGLLp56 3T-GST 20mer GACCGCGAGTGGGTTGCACAAGGCCCTTAATTTTCCAATAA CloningCCTAGTATAGGGAGCTCCG ^(a)Nucleotides used to generate restriction sitesfor cloning are underlined.

C. Peptide Aptamer Screens

The pJM-1 library (Colas et al. (1996) Nature 380:548-550.) wasamplified by transforming 5 μg plasmid DNA into 1×10¹⁰ electro-competentE. coli DH10B cells (Invitrogen) and stored at −80° C. in 40 mL aliquotscontaining 5×10⁸ UFC/mL (Geyer and Brent (2000) Methods Enzymol.328:171-208). Plasmid DNA was extracted using a QIAfilter plasmid maxikit according to the manufacturer's protocols (QIAGEN), Saccharomycescerevisiae strains EGY48 (MAT his3 trp1 ura3-52 leu2::LexA6op-) andEGY191 (MAT his3 trp1 ura3-52 leu2::LexA2op-LEU2) were used for thelibrary screens (Estojak et al. (1995) Mol. Cell. Biol. 15:5820-5829).Plasmid DNA (50 μg) from the library was transformed into the baitstrains containing the lacZ, reporter plasmid pSH18-34 (Invitrogen;Estojak et al. (1995) Mol. Cell. Biol. 15:5820-5829; Golemis and Brent(1992) Mol. Cell. Biol. 12:3006-3014.) and the corresponding baitplasmids, Transformants were plated on synthetic dropout medium lackinghistidine, tryptophan, uracil and leucine and supplemented withgalactose/raffinose (Gal-HWUL) after heat shock and a 4 h incubation at30° C. in liquid medium containing galactose/raffinose and lackinghistidine and uracil (Gal-HU, Golemis and Brent (1992) Mol. Cell. Biol.12:3006-3014). Recovered yeast colonies were also grown in mediumlacking histidine, tryptophan and uracil and supplemented with glucose(Glu-HWU) to repress library expression. Activation of the leucine andβ-galactosidase reporters was confirmed in growth assays (Gal-HWUL) andfilter lift assays (Gal-HWU), respectively (Geyer and Brent (2000)Methods Enzymol. 328:171-208). pJM-1 plasmids containing the selectedaptamers were recovered using the lyticase protocol and QIAGEN miniprepcolumns. The plasmids were transformed into E. coli KC8 strain (ClontechYeast Protocols Manual PT3024-1) and selected on minimal M9 mediumlacking tryptophan. Recovered plasmids were transferred into E. coliDH5α for isolation and retransformed into the yeast baits strains toconfirm specific activation with the TAL1₁₋₃₅₂ and TAL1₁₋₁₃₀ baits andnot with the GUS bait. For these assays, 4 μl droplets of 1×10⁻²dilutions (OD₆₀₀ adjusted to 0.08-0.12) of fresh yeast colonies wereplated onto Gal-HWUL medium and incubated at 30° C. for 3-6 days. Forsequencing, DNA minipreps were performed using the R.E.A.L. Prep 96plasmid kit and a Biorobot 9600 (QIAGEN). Sequencing was performedaccording to the BigDye® Terminator v3.1 method (Applied Biosystems)using a Perkin Elmer Prism 3700 96-capillary automated DNA sequencer.

D. Replication Interference Assays.

Protoplasts were isolated from Nicotiana tabacum (BY-2) suspensioncells, electroporated and cultured according to published methods(Fontes et al. (1994) J. Biol. Chem. 269:8459-8465). For the replicationinterference assays (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122),replicon DNA (2 μg) containing a partial tandem copy of TGMV A(pMON1565; Orozco and Hanley-Bowdoin. (1996) J. Virol. 270:148-158) wascotransfected with a plant expression cassette (40 μg). Viral DNAaccumulation was monitored by either hybridization or semi-quantitativePCR. For the hybridization assays, total DNA was extracted 48 hpost-transfection, digested with DpnI and XhoI, resolved on 1% agarosegels and probed with a ³²P-labeled DNA corresponding to TGMV A.Double-stranded viral DNA accumulation was quantified by phosphorimageranalysis in a minimum of three independent experiments.

For semi-quantitative PCR assays, BY-2 cells were harvested 36 hpost-transfection and lysed by vortexing using 50 μL of glass beads in400 μL lysis buffer (50 mM Tris-HCl pH 7.6, 100 mM NaCl, 50 mM EDTA,0.5% SDS). The lysates were cleared by centrifugation at 14,000 g for 5min and extracted using a QIAprep Spin Miniprep Kit according to themanufacturer protocols (QIAGEN). Total DNA was quantified by A260, andidentical amounts were digested overnight with DpnI and subjected to PCRanalysis using primers LLp1 and LLp2 (Table 3) for the TGMV A replicon.The amount (12.5-200 ng) of total DNA in the reactions was titrated foreach experiment. pMON721 plasmid DNA (1 μg), which does not contain TGMVsequences (Lanahan et al. (1994) Plant Cell 6:521-530) was added to eachPCR reaction as an internal control and amplified with primers LLp39 andLLp40 (Table 3). Bands were quantified using ImageJ software (Abramoffet al. (2004) Biophotonics International 11:36-42; Rasband, W. S. (2005)ImageJ. National Institutes of Health). PCR efficiency was standardizedbetween reactions as a ratio of the band intensities corresponding toTGMV A DNA and the pMON721 control. Relative replication was determinedas the ratio of the normalized intensity of each reaction versus thenormalized intensity detected for protoplasts transfected with TGMV Areplicon DNA and the empty expression cassette pMON921.

D. Sequence Alignments.

For each experimental database, the amino acid content of the peptide20-mers was computed using a script aminocounter.pl that was coded usingBioPerl Modules (Stajich et al. (2002) Genome Res. 12:1611-1618). Basedon this information, 100 random databases of equivalent size and contentwere generated using the Perl script ranPEP.pl (Stajich et al. (2002)Genome Res. 12:1611-1618). The random and experimental peptide databaseswere formatted using NCBI formatdb.exe, and pairwise alignments wereperformed using the NCBI Basic Alignment Search Tool (BLASTP 2.2.10;Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919)with a modified BLOSUM62 matrix (Altschul et al. (1997) Nucleic AcidsRes. 25:3389-3402). The modified matrix removed the stringent gaprestriction and included similarities based in amino acid hydrophobicityand charge. An E value of 20 (scores of 10 bits or more) was used ascutoff for the alignments, which were recorded as the number of peptideswith hits and the sum of hits for all 20-mers in each database. Thesefrequencies were used to calculate the expected or observed mean and thestandard error of the mean for each database, which were compared inone-way T-tests in JMP 5.1 (SAS). The pairwise alignments of theexperimental databases were analyzed further using the Vector NTI AlignXmodule (Invitrogen) to identify potential consensus motifs.

Example 3 Yeast Dihybrid Library Screen

Geminivirus replication proteins contain several conserved motifs thatare essential for function. To develop new approaches to study andmodulate these proteins, the pJM-1 library for interacting peptidesusing the well-characterized TGMV replication protein designated here asTAL1 was screened. The pJM-1 library encodes E. coli thioredoxin (TrxA)with 2.9×10^(a) random 20-mer peptides in its active site (Colas et al.(1996) Nature 380:548-550). The TrxA-peptides are fused to the SV40nuclear localization signal, the E. coli B42 activation domain (AD) andthe hemagglutinin epitope tag and are expressed from the yeast Gallpromoter (Colas et al. (1996) Nature 380:548-550). For the screens, twotypes of TAL1 bait plasmids (FIG. 2A) were constructed. TAL1₁₋₃₅₂encodes the full-length TAL1 protein fused to the E. coli LexA DNAbinding domain (DBD). TAL1₁₋₁₃₀ specifies a LexA DBD fusion with thefirst 130 amino acids of TAL1, which includes the conserved Motifs I, IIand III (FIG. 1). A bait (CaAL1₁₋₃₄₉) containing the LexA DBD fused tothe full-length coding sequence of the Cabbage leaf curl virus (CaLCuV)replication protein designated here as CaAL1 was also constructed. GUSencoding a DBD: β-glucuronidase fusion as a negative control wasemployed.

The advantages of the oligomerization properties of TAL1 (Orozco et al.(2000) J. Biol. Chem. 275:6114-6122) were used to test the functionalityof our full-length AL1 baits (FIG. 2A). Yeast carrying the TAL1₁₋₃₅₂bait and AD:TAL1₁₋₃₅₂ prey plasmids were able to activate the Leureporter in the presence of galactose (Gal-HWUL plates, FIG. 2B-1),consistent with the ability of full-length TAL1 to form oligomers.Transformants carrying the CaAL1₁₋₃₄₉ bait and AD:TAL1₁₋₃₅₂ preyplasmids also grew (FIG. 2B-7), indicating that the heterologous CaAL1protein is able to interact with TAU. In contrast, no growth wasobserved for the TAL1₁₋₁₃₀ bait (FIG. 2B-3), which lacks theoligomerization domain (Orozco et al. (1997) J. Biol. Chem.272:9840-9846), or the GUS bait (FIG. 2B-5) in cotransfection assayswith the AD:TAL1₁₋₃₅₂ prey plasmid. None of the baits interacted withthe AD:Jun (FIGS. 2B-2, 4, 6 and 8) or the AD:TrxA-GST prey plasmids(data not shown). All of the bait/prey combinations also failed to growon selective medium supplemented with glucose (Glu-HWUL plates, FIG.2B). The presence of the bait and prey plasmids in the transformants wasverified by growth on Glu-HWU plates. Similar results were obtained whenactivation of the LacZ reporter was used to detect interactions (datanot shown). Together, these data established the specificity of theTAL1₁₋₃₅₂ and CaAL1₁₋₃₄₉ interactions. These results also verified thatinteraction is dependent on galactose induction of prey plasmidexpression, and that none of the bait plasmids autoactivate the yeastreporters.

A two-step transformation protocol was used for the two-hybrid screensof the pJM-1 library (Geyer and Brent (2000) Methods Enzymol.328:171-208). The yeast strain EGY48 was first co-transformed with theLacZ reporter pSH18-34 and the TAL1₁₋₃₅₂ bait plasmid. The recoveredbait strains were transformed with 50 μg of the library DNA. A total of5×10⁶ colonies were plated on selective media (Gal-HWUL) in 2transformation events, resulting in the recovery of 350 colonies. Thesecolonies were transferred to Glu-HWU plates, grown for 2 days to repressthe library expression and re-evaluated for induction of the Leu andLacZ reporters on HWUL and HWU media supplemented with either galactoseor glucose. Prey plasmids were recovered from 350 colonies that grewonly in the presence of galactose. Retransformation assays using baitstrains carrying TAL1₁₋₃₅₂ or GUS verified the specificity ofinteraction for 170 of the recovered plasmids, 40 of which weresequenced. Eleven TrxA-peptides with unique sequences and no stop codonsor frameshifts were selected for further analysis (Table 4).

TABLE 4 Aptamers isolated in screens with TAL1₁₋₃₅₂ Yeast ExpressionAptamer Peptide sequence plasmid^(a) cassette^(b) FL-1PLSGRQGVHLYFLLLMPA B1118-001 pNSB1138 FL-7 FAVEYGSQGWGLWYCVWLDLB1118-007 pNSB1141 FL-18 FQSRMGGGSGVVNAKLWAKE B1118-018 pNSB1137 FL-19VASRDSGAWRELHSFLNFAS B1118-019 pNSB1135 FL-41 YYMALLYSQCPTVVLFRMTTB1118-041 pNSB1139 FL-42 DFVCLCLFACTSDLSAFRVC B1118-042 pNSB1136 FL-57TAFRWDMFWMHTSGTWRKP B1118-057 pNSB1143 FL-60 FASGSGEPVGLGLGSPLEKLB1118-060 pNSB1144 FL-70 VYDSALCLVVGRCGLIRCR B1118-070 pNSB1134 FL-90LVWASM B1118-090 pNSB1142 FL-99 LHESCWGWAGDSSPQGVLAG B1118-099 pNSB1140^(a)The yeast plasmids were cloned into pJM-1 with carbenicillinselection. ^(b)The plant expression cassettes were cloned into pMON921with carbenicillin selection.

To facilitate the identification of TrxA-peptides that inhibit theessential DNA binding and cleavage activities mediated by the N-terminusof AL1 (Orozco et al. 1997), the pJM-1 library with the TAL1₁₋₁₃₀ bait(FIG. 2A) was also screened. The screens were performed as describedabove for the TAL1₁₋₃₅₂ bait except for the use of the stringent yeaststrain EGY191 to ensure the identification of high affinity interactions(Golemis and Brent (1992) Mol. Cell. Biol. 12:3006-3014). In thisexperiment, 597 positive candidates were isolated from a screen of 2×10⁷yeast colonies. Interaction with TAL1₁₋₁₃₀ was confirmed for 287 yeastcolonies displaying activation of both the Leu and LacZ reporters. Preyplasmids were recovered and sequenced for 130 colonies, out of which 88unique TrxA-peptides were selected (Table 5).

TABLE 5 Aptamers isolated in screens with TAL1₁₋₁₃₀ Yeast ExpressionYeast Expression Aptamer Peptide sequence plasmid^(a) cassette^(b)Aptamer Peptide sequence plasmid^(a) cassette^(b) A-3GFRAPGLSPTRPSCLICSTL B1162-3 pNSB1228 A-114 VRSHRRYQRNWEPVVSWFSSB1162-114 pNSB1279 A-5 NECLICHMLGIREFGLSA B1162-5 pNSB1229 A-115WCGPQVSARCK B1162-115 pNSB1253 A-6 GTLWRRCASSWAFPPDCPSA B1162-6 pNSB1230A-116 SCDEAFDAASVASELFCQPY B1162-116 pNSB1280 A-8 RRALRHCTGCMLSQRLGTALB1162-8 pNSB1258 A-117 ARMALSLREWEYLFFKDAPSGPGL B1162-117 pNSB1228 A-9HSMHSCSVGRCLVDVKVVVS B1162-9 pNSB1231 QGLSLASRLNLVILRGYG A-12WMVCAGCGALRTRQVTLHPG B1162-12 pNSB1232 A-119 RSYGGGEIPSVTMHCWIHCDB1162-119 pNSB1281 A-15 GGFVPMRLCTCLLIVRLFI B1162-15 pNSB1264 A-123SSSRWVPFALQDPLFSSDDW B1162-123 pNSB1282 A-16 VPQPLNCDLCVLMGGASSSRB1162-16 pNSB1233 A-124 YLWSSKMDEWVAMDDVYAAC B1162-124 pNSB1283 A-18RRDYRKFFALNCQLCRLTVT B1162-18 pNSB1234 A-127 TWGLVCTGTGWGLLDTVVRAB1162-127 pNSB1284 A-22 CRTRGCGCHLCRMLSQFTGG B1162-22 pNSB1235 A-129VYEWGDVLCGGSMAIQWGL B1162-129 pNSB1254 A-25 MRLGKGWNLMFLEEVSVLDAB1162-25 pNSB1323 A-130 ASNGEIAYCVEQAMLLLCFH B1162-130 pNSB1285 A-27RDPQLGQVAQTWGCRLCLLE B1162-27 pNSB1259 A-131 ELIVHEWPLILSRVGRIVLB1162-131 pNSB1255 A-30 LVSESCGSWFCLCPWEVLNW B1162-30 pNSB1263 A-132GRVQLEILVSEAEEGVSPFL B1162-132 pNSB1286 A-40 LQYSWNLYSVASFKTRRVSSB1162-40 pNSB1236 A-135 RDAEWQDVLGRARAVHLRGR B1162-135 pNSB1287 A-41RLQESSIDLTPGIYLGMDFV B1162-41 pNSB1265 A-136 GLKWKSDNGCVYVSFMRGGVB1162-136 pNSB1288 A-46 CYMEVEGRPRRWADSFFVAW B1162-46 pNSB1262 A-137SSSPVPYSGGTCNLCSMRMW B1162-137 pNSB1289 A-49 SESFVCKTCHMLRVSDAVGAB1162-49 pNSB1260 A-140 EWEDPQYAGWELFSISDLVH B1162-140 pNSB1256 A-50MHVSLVFPWRLTGHIQQYKV B1162-50 pNSB1261 A-141 PMVRTEWPLCAIIPLSMLYQB1162-141 pNSB1290 A-51 GRCNLQGMSFMGVGRSVWFE B1162-51 pNSB1237 A-143RAGWHERVRQWWAIECTLEV B1162-143 pNSB1291 A-53 VVGGSLRDEWKWWREGRSLPB1162-53 pNSB1267 A-145 SVRCWYVLRCSFLVGSGSSV B1162-145 pNSB1292 A-59AKDVERGAGGKIKACELCRL B1162-59 pNSB1268 A-146 RSCVLCAYGSRTFNGSYLLFB1162-146 pNSB1257 A-63 VETFKARARQTPSCDLCPKT B1162-63 pNSB1238 A-147GRGGCMLCDVDGSSAWLHTEGRLT B1162-147 pNSB1266 A-64 TELWWADFAKMHMEGGKGMCB1162-64 pNSB1239 GPITSQQCLSFQYLGNGEFIDG A-67 RHRCTSRAPRQWFRPHRDSPB1162-67 pNSB1269 A-149 TLETLDMGNPLYTCVLMDWM B1162-149 pNSB1324 A-69RYRVSAGPLCSLCSLWGSVG B1162-69 pNSB1240 A-150 LVMGWRSEVSSLQGKTGTGGGPTLB1162-150 pNSB1293 A-71 EEGLAAITHTWLTMCFAAGL B1162-71 pNSB1241RKCQLCRGSRYTLKYYPC A-73 AAFLESVRSYWSRFVRHVQG B1162-73 pNSB1242 A-153RPGCPFCTSWRCG B1162-153 pNSB1294 A-75 RAMCDKDKSVCSILALYVQV B1162-75pNSB1243 A-155 FCPECQMVAGAEDGDAIDLQ B1162-155 pNSB1295 A-80CWWLREIGTFRCVTLQHVAG B1162-80 pNSB1244 A-158 RRCMLCTSDKPGGDQGALNMB1162-158 pNSB1296 A-82 FESAWSTLMGAMTPMVLDET B1162-82 pNSB1270 A-159LWGGGTAWDFFVWGEDSAC B1162-159 pNSB1297 A-83 QALVVSPETFLCLEALGVNSB1162-83 pNSB1271 A-160 GMSGRIPEPDDWVVLFITGC B1162-160 pNSB1298 A-84GGRQTEPSLTLLADLTLLLS B1162-84 pNSB1272 A-161 GGTNALLQKVFFGEVGVASMB1162-161 pNSB1299 A-86 GSRAELSAPEVAWLLFCTPG B1162-86 pNSB1245 A-164ECCLFPIFAMADSFPCPSPV B1162-164 pNSB1300 A-87 RYSAVCRDCYEGHGRGLWYMB1162-87 pNSB1273 A-165 MLEGPLDQGLMMGTCCWECS B1162-165 pNSB1301 A-89GGWLVTIVEGPLAICCLRDD B1162-89 pNSB1274 A-166 TPSVTWAEWCSCVFCRDASB1162-166 pNSB1302 A-90 PSIESGWVGDQAVAPCDLSV B1162-90 pNSB1275 A-167SWWWANNSLCREWEFAC B1162-167 pNSB1303 A-91 TWGAWKRDIVLVSEIGFTWG B1162-91pNSB1246 A-168 WNMLAFGGALVASGLLRGWE B1162-168 pNSB1304 A-94RLGGGRPKLWHFSPNLMAGF B1162-94 pNSB1247 A-169 DKCDDVEPFLWWGQQCFFDVB1162-169 pNSB1305 A-97 ERVHVCFSRKCTALSVDSSV B1162-97 pNSB1248 A-170GSPSRISYTCLSPDVTLLFL B1162-170 pNSB1306 A-99 RERGGDDYRRMMHPGAASGPB1162-99 pNSB1249 A-172 MGIEACSITECTSQHCNEVA B1162-172 pNSB1307 A-100RLVVGCEWRIGCSTGSGPRG B1162-100 pNSB1250 A-173 CLDNLCWELGGGFPVILIHCB1162-173 pNSB1308 A-101 ASLIGVGIASMHGMQTDGIY B1162-101 pNSB1251 A-174HVHGSCPSMGWSSNSWCSVF B1162-174 pNSB1309 A-108 VGLMEWAVWSLEVREKLYSCB1162-108 pNSB1276 A-175 PLELEFAVCGCSWLVALDWS B1162-175 pNSB1310 A-109VLGRLGGAGGCSLCDQLEAL B1162-109 pNSB1277 A-176 AWDSESLATVVASVMPWPYPTB1162-176 pNSB1311 A-110 IWINPNGLWWTKVGLNPYAV B1162-110 pNSB1252 A-177TGCHYKGARCCRLTWDVLIL B1162-177 pNSB1312 A-112 RHESALHKSCELCYCPWKVCB1162-112 pNSB1278 ^(a)The yeast plasmids were cloned into pJM-1 withcarbenicillin selection. ^(b)The plant expression cassettes were clonedinto pNSB1226 with kanamycin selection.

Because these TrxA-peptides were selected for binding to a truncatedTAL1 protein that does not oligomerize, interaction with full-lengthTAL1 was investigated. FIG. 3B shows that yeast co-transformed with eachof the 88 TrxA-peptides in the presence of either TAL1₁₋₁₃₀ or TAL1₁₋₃₅₂baits grew on in Gal-HWUL medium. Co-transfection with the negativecontrol bait GUS did not induce the Leu reporter and no growth occurred(FIG. 3B). No growth was seen on plates lacking leucine but supplementedwith glucose (FIG. 3C). Growth on Glu-HWU medium confirmed the presenceof the prey and bait plasmids in all of the transfections (FIG. 3D).Based on these results, it can be concluded that the 88 TrxA-peptidesinitially selected for interaction with TAL1₁₋₁₃₀ also bind toTAL1₁₋₃₅₂.

Example 4 Aptamer Interference with Viral DNA Replication

After the initial screening, a subsequent aptamer library screen wasperformed to identify aptamer sequences that specifically bind to theN-terminal domain of Rep and impact Rep function during viralreplication.

A. Binding of TrxA-Peptides to TGMV AL1 and Interference with Viral DNAReplication.

The 11 FL-TrxA-peptides (Table 4) selected in the screen using theTAL1₁₋₃₅₂ bait were subcloned into a plant expression cassettecontaining the CaMV35S promoter with a duplicated enhancer and the rbcSE9 terminator. The constructs were co-transfected into tobaccoprotoplasts in the presence of a replicon plasmid containing a partialtandem copy of TGMV A that supports the release of unit length viral DNA(FIG. 4A). The TGMV A DNA, which encodes TAL1 and its replicationaccessory factor AL3 (FIG. 4A), replicates autonomously and accumulatesto high copy number in plant cells (Orozco and Hanley-Bowdoin. (1996) J.Virol. 270:148-158). To determine whether expression of theFL-TrxA-peptides quantitatively impacts TGMV A DNA accumulation as anindicator of altered TAL1 activity, total DNA was isolated 48 h posttransfection, and the levels of double-stranded TGMV A DNA were examinedby DNA gel blot hybridization. Nine of the FL-TrxA-peptides had nodetectable effect on viral DNA accumulation (data not shown). Incontrast, cells containing the FL-42 (FIG. 4B, lane 3) and FL-60 (lane4) cassettes only accumulated about 25% of the levels detected in atransfection containing an empty expression cassette (lane 2). The levelof viral DNA detected in the presence of FL-42 and FL-60 was similar tothat seen for FQ118 (FIG. 4B, lane 1), a strong trans-dominant negativemutant of TAL1 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122).These results indicated that two of the FL-TrxA-peptides interfere withthe ability of TAL1 to support viral DNA replication.

The 88 N-TrxA-peptides were also tested in replication interferenceassays. For these experiments, a semi-quantitative PCR protocol wasdeveloped to facilitate the analysis of a large number of expressioncassettes. The assay was based on primers that distinguish the inputreplicon cassette and nascent viral DNA by size and DpnI sensitivity.Primers LLp1 and LLp2 (Table 3 and FIG. 4A) amplify a 4.9 Kb DNA fromthe replicon cassette and a 1.2 Kb product from the released TGMV Acomponent (FIG. 4C, lane 1) in DNA extracts from E. coli cells carryingthe replicon cassette plasmid. Even though the replicon cassette is theprevalent form in E. coli (data not shown), it amplified less,efficiently than the released TGMV component because of its large size.The production of both products is sensitive to DpnI digestion (FIG. 4C,lane 2) and resistant to MboI digestion (lane 3), indicative of an E.coli Dam-methylated template. Interestingly, the same results (FIG. 4C,lanes 4-6) were obtained for the mutant TGMV A replicon cassette with aframe shift mutation at TAL1 amino acid position 120 (Elmer et al.(1988) Nucleic Acids Res. 16:7043-7060).

The amplification strategy was also tested with DNA extracted fromtobacco cells co-transfected with various TGMV A replicon and expressioncassettes. It was then determined whether the input and nascent viralDNA can be distinguished by comparing DNA samples isolated from cellstransfected with the wild type TGMV A replicon cassette or the mutantAL1 cassette. The 1.2 Kb product (top band) was produced when uncut andMboI-digested DNA from cells transfected with both cassettes wasamplified (FIG. 4D, lanes 1-3 and 10-12, top and bottom). In contrast,the 1.2 Kb product was only amplified from DpnI-treated DNA from cellswith the wild type cassette (FIG. 4D, lanes 1-3, middle) but not themutant cassette (lanes 10-12, middle). This result demonstrated thatresidual Dam-methylated E. coli DNA can be quantitatively removed byDpnI digestion, thereby allowing the detection of nascent DNA by PCR.Subsequent verification that replication interference can be monitoredby PCR was obtained by showing that the level of the 1.2 Kb PCR productis reduced in cells carrying a TAL1 dominant negative mutant (FQ118)expression cassette (FIG. 4D, lanes 7-9) relative to cells with theempty (lanes 1-3) or TrxA-GST (lanes 7-9) cassettes. This difference wasnot apparent in uncut or MboI-treated DNA because of the presence ofintact E. coli input DNA (FIG. 4D, lanes 1-9, upper and lower). Similarresults were seen for the three biological replicas for eachtransfection condition. Together; these results establish that the PCRassay can be used to monitor viral DNA accumulation in a reproducible,semi-quantitative manner.

Expression cassettes corresponding to the 88 N-TrxA-peptides (Table 5)were transfected into tobacco protoplasts with the wild type TGMV Areplicon cassette. Total DNA was isolated 36 h after transfection andanalyzed in replication interference assays using the semi-quantitativePCR method. Because of the high number of samples, the N-TrxA-peptideswere initially analyzed in triplicate in 3 separate experiments. 35N-TrxA-peptide cassettes that reduced viral DNA accumulation relative tothe empty expression cassette were selected. The selected cassettes werethen assayed in a single transfection experiment (FIG. 5), with 31 of 35showing statistically significant interference activity (p<0.05 in aone-tailed Students T-test). The experiment also included the FQ118 andTrxA-GST expression cassettes as positive and negative controls,respectively. The N-TrxA-peptides were classified as weak (50-65%),moderate (25-50%) and strong (<25%) interferers (FIG. 5, dotted lines)relative to the control transfection with an empty cassette (100%). TenN-TrxA-peptides showed strong interference (black bars), fourteenexhibited moderate interference (grey bars), and seven were weakinterferers (white bars). In total, fourteen aptamers displayedinterference activity that was greater or equal to FQ118. TrxA-GST didnot impact viral DNA accumulation, indicating the TrxA sequences per sedo not contribute to interference.

B. Binding of N-TrxA-Peptides to CaAL1 and Interference with Viral DNAReplication.

Motifs I, II and III in TAL1₁₋₁₃₀ (FIG. 1) are conserved in allgeminivirus replication proteins (Ilyina and Koonin (1992) Nucleic AcidsRes. 20:3279-3285; Koonin and Ilyina (1992) J. Gen. Virol.73:2763-2766). Hence, it was determined whether peptides that bind toTAL1₁₋₁₃₀ and inhibit TGMV replication are also able to interact with anAU protein from a heterologous geminivirus. Accordingly, experimentswere conducted using CaLCuV AL1 protein because it only shares 42%identity and 58% similarity with TAL1 across the first 130 amino acids.Full-length CaAL1 fused to the LexA DBD (CaAL1₁₋₃₄₉) was used as bait inyeast two-hybrid assays with the 31 N-TrxA-peptides that displayedreplication interference activity (FIG. 5). All of the peptides werepositive for interaction with CaAL1₁₋₃₄₉ in growth assays (FIG. 6B,right). The prey control did not interact with any of the baits (FIG.6). Comparison of the interactions with TAL1₁₋₁₃₀, TAL1₁₋₃₅₂ andCaAL1₁₋₃₄₉ baits on LacZ plates suggested that interaction is strongerwith TAL1₁₋₁₃₀ than with the two full-length AU baits (data not shown).

Example 5 Pairwise Alignments of Peptide Aptamers

To determine whether the peptide aptamers of the present invention arerandom or related in view of their selection for binding to TAL1 the20-mers were grouped into three database corresponding to the 88selected for binding to TAL1₁₋₁₃₀ (All), the 31 positive for replicationinterference (Interfering), and the 57 negative for interference(Non-interfering), and their sequences were compared in pairwisealignments using BLASTP. The score values of the alignments were low(data not shown) because of the short length of the peptide sequences(20 amino acids). To address the possibility that the alignments wereproduced by chance, 100 sets of three random databases of the same sizeand amino acid content as the N-TrxA-peptide were compared using BLASTP.The distribution of the frequency of hits was analyzed for each databaseand used to determine the expected mean and standard error of the meanfor random sequences.

The frequency distribution of the 100 databases comprising the 88 random20-mers is shown in FIG. 7A. The left panel represents the expecteddistribution of peptides with at least one hit while the right panelshows the frequency distribution of the total number of hits for all the20-mers in the database. The expected means of the two distributions (54and 101) are lower than the observed means (67 and 221, respectively)for the All database (FIG. 7B). The observed means for the Interferingand Non-interfering databases are also higher than the expected meanscalculated using the corresponding random databases (FIG. 7B). Theobserved and expected means of all three databases differed by at leasttwo standard deviations and gave p<0.0001 in one-way Students T-tests.These results established that even though N-TrxA-peptides were derivedfrom a random library, their sequences are not random.

Inspection of the BLASTP alignments revealed that some pairs containedcommon sequences. Similar pairs were grouped and compared using theVector NTI AlignX module. A total of 18 groups containing four or moresequences were identified among the 88 N-TrxA peptides. The putativemotifs were filtered using four criteria—[1] include at least fivemembers, [2] members interact with CaAL1, [3] contain amino acidstypically involved in protein-protein interactions (Bogan and Thorn(1998) J. Mol. Biol. 280:1-9; Glaser et al. (2001) Proteins 43:89-102),and [4] related to a plant protein. Seven motifs that satisfied at leastthree criteria are shown in FIG. 8A. The sequence alignments are shownin FIGS. 9 and 10. Motifs 1, 4, 20, 25 and 27 consist primarily ofinterfering peptides, while Motif 28 is composed of mostlynon-interfering peptides. Motif 24 includes 18 members that aredistributed between interfering and non-interfering peptides, all ofwhich contain a core CxLC sequence (FIG. 8). The interfering membersalso include conserved polar and nonpolar residues flanking the coresequence (FIG. 9). These residues occur individually in non-interferingpeptides, but only the interfering peptides contain both sets offlanking residues.

Example 6 Characterization of Additional Peptide Aptamers

To expand the repertoire of aptamers, additional peptides selected inthe dihybrid screen using full-length TGMV Rep were characterized. Thelocations of their binding sites were mapped using a series of baitscorresponding to known Rep functional domains (FIG. 1). The baitscontained Rep₁₋₁₃₀ (DNA binding, cleavage and ligation), Rep₁₀₁₋₁₈₀ (C3and pRBR binding), Rep₁₃₀₋₁₈₀ (GRIK, CRIMP, PCNA and Ubc9 binding andoligomerization), and Rep₁₈₀₋₃₅₂ (helicase) and were designated as FL,NT, PI, OL and CT, respectively. Aptamers were further tested forbinding to full-length CaLCuV Rep.

The results of the yeast two-hybrid experiments are summarized in Table6. The peptide sequences and binding properties of 99 Trx-aptamers thatbound to full-length TGMV Rep and do not contain a frameshift or stopcondon in their coding sequence are shown in Table 7. A comprehensivelisting of peptide sequences according to some embodiments of thepresent invention is provided in Table 8.

TABLE 6 Results summary TGMV baits TGMV Rep CaLCuV Rep FL 22 22 FL, NT 76 FL, PI 8 4 FL, OL 12 10 FL, CT 7 7 FL, NT, PI 7 7 FL, NT, OL 3 3 FL,NT, CT 5 4 FL, PI, OL 5 4 FL, OL, CT 11 10 FL, NT, PI, OL 1 1 FL, NT,PI, CT 2 1 FL, NT, OL, CT 5 4 FL, NT, PI, OL, CT 3 3

TABLE 7 Binding properties and sequences of individual peptide aptamersAptamer name TGMV Rep CaLCuV (FL#) bait* Rep bait* peptide sequence 5 FLYES ELLVAHLITPWTSMGRTQAL 73 FL YES HKDRGYANLMLCSLLACFEP 76 FL YESGMLWTWLCDSPQSFVAPRGV 111 FL YES PVCRVGRGLLVQAKLVRAQS 119 FL YESDLEWDGNAYSGCCHCAFSIR 141 FL YES PNCEICYVARRLVLSMEACS 165 FL YESEGLPIDLLTNWHLTCWIALG 177 FL YES GVNLLQRCWGGPVHIFSYLM 184 FL YESCLMHMRFPLGGTWRMNLRAE 190 FL YES LVTALIMSESIVRMNPMYLT 193 FL YESEVERSRMLFNYGGMVASRVA 194 FL YES PWLSVDVTALIVDFLQDFSA 200 FL YESEGGEDSAWDWGSSGGIWCWF 206 FL YES CDVMEWRMMGLGLSKWLRGR 219 FL YESVYYHKECALDSYVRTCWVSG 225 FL YES PSAYEAVETLDMSEVKGLGQ 237 FL YESCEDMREAKVCRTLLAHSFLP 251 FL YES TSWRHRMPTGTDRCCFLVQL 275 FL YESTIDQRMVSLGAIWWSYPRCW 293 FL YES AQRSCWERLWTGQWRRSASD 322 FL YESWGYFGSFVGGVFDVWFSGVA 326 FL YES RNGRNICVLSVCSRFSHFNP 133 FL, NT YESFGVIVTNAASEFTTRVDDSC 199 FL, NT YES FNARALACKCDRGILILSQP 214 FL, NT YESIYDYTWAEEQGYVWRPAGGA 227 FL, NT YES LARCLCEIVGSCISYSNLPI 239 FL, NT YESRPWSSDTSVWWDGLFGMNYS 252 FL, NT NO RPWSSDTSVWWDGLFGMNYS 281 FL, NT YESTEACQVVLLGKRSLLPVVAG 52 FL, PI YES LARQPGKFIELPVLIRFATSGPLLQSNSSSGHWLSDEHWTRR 180 FL, PI YES VDSIDKGEALVSLWGWHVQI 189 FL, PI YESLVRWSYTCSVLVGLRDSLDS 278 FL, PI NO PPWTKRPLTSGGVRGELWVW 280 FL, PI NORVDSLGIKLDKSTLVTVHVV 294 FL, PI NO VSWSAAGRGYVFMYRWSPRC 325 FL, PI NOSRSDLWVSWCRNLLDGQSWS 381 FL, PI YES MLGIWRVLHEMVVPLKLGVD 58 FL, OL NOGKLTDTTRISVCCICVSVLD 72 FL, OL YES MDRREDLRSLLVLTLSDARG 100 FL, OL YESPLLLLAADSVELQRVLARR 107 FL, OL YES AMTYRAAWSPPPWGVLGIWH 154 FL, OL YESRLTVLEAAVVLWGWSLFQVP 197 FL, OL YES VIVDFLSTGVSTGEVRGGIV 221 FL, OL YESTLHTNRFCFRWVPALDSVTT 256 FL, OL NO LVTALIMSESIVRMNPMYLT 258 FL, OL YESGVNNGGTNDPEGVSEASWIP 300 FL, OL YES FGVIVTNAASEFTTRGYDSC 304 FL, OL YESSRSDLWVSWCRNLLDGQSWS 382 FL, OL YES CPDCPLSSVLRTATTAFFGG 121 FL, CT YESQSVRKMPMFWPLAGEVWCRPLGFR 181 FL, CT YES PPWTKRPLTSGGVRGELWVW 196 FL, CTYES ISSYLWWSEYCRPGSAMGDV 218 FL, CT YES RVWTFFVREAALELPSRDTL 222 FL, CTYES LNPWEGEWTRWDVFRVLGEF 265 FL, CT YES GIVSKQGADEGMLEIFASSW 292 FL, CTYES AQRSCWERLWTGQWRRLPLM 8 FL, NT, PI YES DEQESVCRSCKCRYVDNWLE 25FL, NT, PI YES HKDRGYANLMLCSLLACFEP 117 FL, NT, PI YESSFVVQSFLGGKSIFNGPFAD 120 FL, NT, PI YES LGAPLLRCMVHHAMMVGEGY 135FL, NT, PI YES RPWSSDTSVWWDGLFGMNYS 232 FL, NT, PI YESSFATANSEQVLRDMLLLASH 364 FL, NT, PI YES TGSGLTPCLHCRVQFQRSYL 216FL, NT, OL YES QMNAAPPARSCADTWSLLLF 229 FL, NT, OL YESSGYPKMVWGEGPMLLDWKFV 246 FL, NT, OL YES WCSMCSVLRAFNCPYFCPWL 14FL, NT, CT YES VGGMPPLPWYEPVGLVWSCM 21 FL, NT, CT YESGKLTDTRISVCCCICVSVLD 167 FL, NT, CT YES SFMMRLLRTGEMQFQADCVGGPIPLKSPRALSLYNWGLLLWV 202 FL, NT, CT YES LLYPSLTLTLWRWLFADEGC 247FL, NT, CT NO PWIFDRSVVCEEREAPRRHL 6 FL, PI, OL YESTNELPLTIVTDDVSQLVISRGP ARHLYELMPEMLVLRSARLT 334 FL, PI, OL YESGVLFTFKKYPQGLSCTTSYG 341 FL, PI, OL NO LLACSVYWLWQRPCDGCLFM 365FL, PI, OL YES TRMHSLCSGFCVICMGGPRV 380 FL, PI, OL YESRNGRNICVLSVCSRFSHFNP 109 FL, OL, CT YES GMLWTWLCDSPQSFVAPRGV 116FL, OL, CT YES EVVETAEVYWSCGDWSCEGW 179 FL, OL, CT YESCAMCLDVFGWSASHWGGFTV 220 FL, OL, CT YES FNARALACKCDRRILILSQP 245FL, OL, CT YES GRFGQGQCYQVADSTYWTFGPG GPRKCEREPAGWSDTGWVC 254 FL, OL, CTYES RPWSSDTSVWWDGLFGMNYS 262 FL, OL, CT YES LLACSVYWLWQRPCDGCLFM 302FL, OL, CT NO SFSSLFLAWLMQTGQEAGTV 312 FL, OL, CT YESHPYLITDIISMYRSPWSVPA 352 FL, OL, CT YES VVQNVRGWLVYCCADFHTYV 355FL, OL, CT YES. LRGVSPWLQSFVSIAVQSCK 313 FL, NT, PI, OL YESRDALTNIGRSICALLLVLCK 153 FL, NT, PI, CT YES PNCEICYVARRLVLSMEACS 223FL, NT, PI, CT NO QMFWFTDSEGKPGFCTFYGF 110 FL, NT, OL, CT YESALKDEPFCDLPMVLVSWWRG 208 FL, NT, OL, CT YES DLEWDGNAYSGCCHCAGSIR 257FL, NT, OL, CT YES LLACSVYWLWQRPCDGCLFM 328 FL, NT, OL, CT NOGGSDERYFWYQSFSSCAYEW 434 FL, NT, OL, CT YES RGRMEADKSFDSTCLRCGCS 236FL, NT, PI, OL, CT YES VGPLIVGPPGMEMTANSWSC 242 FL, NT, PI, OL, CT YESDLRLPVYSEWVRVYSSDAWM 293 FL, NT, PI, OL, CT YES AQRSCWERLWTGQWRRLPLM*TGMV baits FL—full length (aa 1-352) NT—N-terminus (aa 1-130)PI—protein interaction (aa 101-180) OL—oligomerization (aa 130-180)CT—C-terminus (181-352) CaLCuV bait full length (aa 1-347)

TABLE 8 Listing of peptide sequences SEQ ID NO. Research NamePeptide sequence 1 Motif 3 VRDYILKEPL 2 Motif 3 VKSYVDKDGD 3 Motif 3VKEYIDKDGV 4 Motif 3 VNSYVDKDGD 5 Motif 3 NKEYCSKEGH 6 Motif 3NLTYVSKIGG 7 Motif 3 AQLYAMKEDS 8 Motif 3 ARSYCMKEDT 9 Motif 1 WxDxxxAW10 Motif 4 MHxxxxxG 11 Motif 20 (L/M)GGxxP 12 Motif 24 (G/S)CxLCxL 13Motif 25 WxxxSLC 14 Motif 27 (S/A)FxxAxVAS 15 Motif 28 WfxVL 16 A-3GFRAPGLSPTRPSCLICSTL 17 A-5 NECLICHMLGIREFGLSA 18 A-6GTLWRRCASSWAFPPDCPSA 19 A-8 RRALRHCTGCMLSQRLGTAL 20 A-9HSMHSCSVGRCLVDVKVVVS 21 A-12 WMVCAGCGALRTRQVTLHPG 22 A-15GGFVPMRLCTCLLIVRLFI 23 A-16 VPQPLNCDLCVLMGGASSSR 24 A-18RRDYRKFFALNCQLCRLTVT 25 A-22 CRTRGCGCHLCRMLSQFTGG 26 A-25MRLGKGWNLMFLEEVSVLDA 27 A-27 RDPQLGQVAQTWGCRLCLLE 28 A-30LVSESCGSWFCLCPWEVLNW 29 A-40 LQYSWNLYSVASFKTRRVSS 30 A-41RLQESSIDLTPGIYLGMDFV 31 A-46 CYMEVEGRPRRWADSFFVAW 32 A-49SESFVCKTCHMLRVSDAVGA 33 A-50 MHVSLVFPWRLTGHIQQYKV 34 A-51GRCNLQGMSFMGVGRSVWFE 35 A-53 VVGGSLRDEWKWWREGRSLP 36 A-59AKDVERGAGGKIKACELCRL 37 A-63 VETFKARARQTPSCDLCPKT 38 A-64TELWWADFAKMHMEGGKGMC 39 A-67 RHRCTSRAPRQWFRPHRDSP 40 A-69RYRVSAGPLCSLCSLWGSVG 41 A-71 EEGLAAITHTWLTMCFAAGL 42 A-73AAFLESVRSYWSRFVRHVQG 43 A-75 RAMCDKDKSVCSILALYVQV 44 A-80CWWLREIGTFRCVTLQHVAG 45 A-82 FESAWSTLMGAMTPMVLDET 46 A-83QALVVSPETFLCLEALGVNS 47 A-84 GGRQTEPSLTLLADLTLLLS 48 A-86GSRAELSAPEVAWLLFCTPG 49 A-87 RYSAVCRDCYEGHGRGLWYM 50 A-89GGWLVTIVEGPLAICCLRDD 51 A-90 PSIESGWVGDQAVAPCDLSV 52 A-91TWGAWKRDIVLVSEIGFTWG 53 A-94 RLGGGRPKLWHFSPNLMAGF 54 A-97ERVHVCFSRKCTALSVDSSV 55 A-99 RERGGDDYRRMMHPGAASGP 56 A-100RLVVGCEWRIGCSTGSGPRG 57 A-101 ASLIGVGIASMHGMQTDGIY 58 A-108VGLMEWAVWSLEVREKLYSC 59 A-109 VLGRLGGAGGCSLCDQLEAL 60 A-110IWINPNGLWWTKVGLNPYAV 61 A-112 RHESALHKSCELCYCPWKVC 62 A-114VRSHRRYQRNWEPVVSWFSS 63 A-115 WCGPQVSARCK 64 A-116 SCDEAFDAASVASELFCQPY65 A-117 ARMALSLREWEYLFFKDAPSGPGLQGLSLASRLNLVI LRGYG 66 A-119RSYGGGEIPSVTMHCWIHCD 67 A-123 SSSRWVPFALQDPLFSSDDW 68 A-124YLWSSKMDEWVAMDDVYAAC 69 A-127 TWGLVCTGTGWGLLDTVVRA 70 A-129VYEWGDVLOGGSMATQWGL 71 A-130 ASNGEIAYCVEQAMLLLCFH 72 A-131ELIVHEWPLILSRVGRIVL 73 A-132 GRVQLEILVSEAEEGVSFL 74 A-135RDAEWQDVLGRARAVHLRGR 75 A-136 GLKWKSDNGCVYVSFMRGGV 76 A-137SSSPVPYSGGTCNLCSMRMW 77 A-140 EWEDPQYAGWELFSISDLVH 78 A-141PMVRTEWPLCATIPLSMLYQ 79 A-143 RAGWHE RVRQWWAIECTLEV 80 A-145SVRCWYVLRCSFLVGSGSSV 81 A-146 RSCVLCAYGSRTFNGSYLLF 82 A-147GRGGCMLCDVDGSSAWLHTEGRLTGPITSQQCLSFQYLGNGEFIDG 83 A-149TLETLDMGNPLYTCVLMDWM 84 A-150 LVMGWRSEVSSLQGKTGTGGGPTLRKCQLCRGSRYTLKYYPC85 A-153 RPGCPFCTSWRCG 86 A-155 FCPECQMVAGAEDGDAIDLQ 87 A-158RRCMLCTSDKPGGDQGALNM 88 A-159 LWGGGTAWDFFVWGEDSAC 89 A-160GMSGRIPEPDDWVVLFITGC 90 A-161 GGTNALLQKVFFGEVGVASM 91 A-164ECCLFPIFAMADSFPCPSPV 92 A-165 MLEGPLDQGLMMGTCCWECS 93 A-166TPSVTWLAEWCSCVFCRDAS 94 A-167 SWWWANNSLCREWEFAC 95 A-168WNMLAFGGALVASGLLRGWE 96 A-169 DKCDDVEPFLWWGQQCFFDV 97 A-170GSPSRISYTCLSPDVTLLFL 98 A-172 MGIEACSITECTSQHCNEVA 99 A-173CLDNLCWELGGGFPVILIHC 100 A-174 HVHGSCPSMGWSSNSWCSVF 101 A-175PLELEFAVCGCSWLVALDWS 102 A-176 AWDSESLATWASVMPWPYPT 103 A-177TGCHYKGARCCRLTWDVLIL 104 FL-1 PLSGRQGVHLYFLLLMPA 105 FL-7FAVEYGSQGWGLWYCVWLDL 106 FL-18 FQSRMGGGSGVVNAKLWAKE 107 FL-19VASRDSGAWRELHSFLNFAS 108 FL-41 YYMALLYSQCPTVVLFRMTT 109 FL-42DFVCLCLFACTSDLSAFRVC 110 FL-57 TAFRWDMFWMHTSGTWRKP 111 FL-60FASGSGEPVGLGLGSPLEKL 112 FL-70 VYDSALCLVVGRCGLIRCR 113 FL-90 LVWASM 114FL-99 LHESCWGWAGDSSPQGVLAG 115 FL-5 ELLVAHLITPWTSMGRTQAL 116 FL-6TNELPLTIVTDDVSQLVISRGPARHLYELMPEMLVLRSARLT 117 FL-8 DEQESVCRSCKCRYVDNWLE118 FL-14 VGGMPPLPWYEPVGLVWSCM 119 FL-21 GKLTDTRISVCCCICVSVLD 120 FL-25HKDRGYANLMLCSLLACFEP 121 FL-52LARQPGKFIELPVLIRFATSGPLLQSNSSSGHWLSDEHWTRR 122 FL-58GKLTDTTRISVCCICVSVLD 123 FL-72 MDRREDLRSLLVLTLSDARG 124 FL-73HKDRGYANLMLCSLLACFEP 125 FL-76 GMLWTWLCDSPQSFVAPRGV 126 FL-100PLLLLAADSVELQRVLARR 127 FL-107 AMTYRAAWSPPPWGVLGIWH 128 FL-109GMLWTWLCDSPQSFVAPRGV 129 FL-110 ALKDEPFCDLPMVLVSWWRG 130 FL-111PVCRVGRGLLVQAKLVRAQS 131 FL-116 EVVETAEVYWSCGDWSCEGW 132 FL-117SFVVQSFLGGKSIFNGPFAD 133 FL-119 DLEWDGNAYSGCCHCAFSIR 134 FL-120LGAPLLRCMVHHAMMVGEGY 135 FL-121 QSVRKMPMFWPLAGEVWCRPLGFR 136 FL-133FGVIVTNAASEFTTRVDDSC 137 FL-135 RPWSSDTSVWWDGLFGMNYS 138 FL-141PNCEICYVARRLVLSMEACS 139 FL-153 PNCEICYVARRLVLSMEACS 140 FL-154RLTVLEAAVVLWGWSLFQVP 141 FL-165 EGLPIDLLTNWHLTCWIALG 142 FL-167SFMMRLLRTGEMQFQADCVGGPIPLKSPRALSLYNWGLLLWV 143 FL-177GVNLLQRCWGGPVHIFSYLM 144 FL-179 CAMCLDVFGWSASHWGGFTV 145 FL-180VDSIDKGEALVSLWGWHVQI 146 FL-181 PPWTKRPLTSGGVRGELWVW 147 FL-184CLMHMRFPLGGTWRMNLRAE 148 FL-189 LVRWSYTCSVLVGLRDSLDS 149 FL-190LVTALIMSESIVRMNPMYLT 150 FL-193 EVERSRMLFNYGGMVASRVA 151 FL-194PWLSVDVTALIVDFLQDFSA 152 FL-196 ISSYLWWSEYCRPGSAMGDV 153 FL-197VIVDFLSTGVSTGEVRGGIV 154 FL-199 FNARALACKCDRGILILSQP 155 FL-200EGGEDSAWDWGSSGGIWCWF 156 FL-202 LLYPSLTLTLWRWLFADEGC 157 FL-206CDVMEWRMMGLGLSKWLRGR 158 FL-208 DLEWDGNAYSGCCHCAGSIR 159 FL-214IYDYTWAEEQGYVWRPAGGA 160 FL-216 QMNAAPPARSCADTWSLLLF 161 FL-218RVWTFFVREAALELPSRDTL 162 FL-219 VYYHKECALDSYVRTCWVSG 163 FL-220FNARALACKCDRRILILSQP 164 FL-221 TLHTNRFCFRWVPALDSVTT 165 FL-222LNPWEGEWTRWDVFRVLGEF 166 FL-223 QMFWFTDSEGKPGFCTFYGF 167 FL-225PSAYEAVETLDMSEVKGLGQ 168 FL-227 LARCLCEIVGSCISYSNLPI 169 FL-229SGYPKMVWGEGPMLLDWKFV 170 FL-232 SFATANSEQVLRDMLLLASH 171 FL-236VGPLIVGPPGMEMTANSWSC 172 FL-237 CEDMREAKVCRTLLAHSFLP 173 FL-239RPWSSDTSVWWDGLFGMNYS 174 FL-242 DLRLPVYSEWVRVYSSDAWM 175 FL-245GRFGQGQCYQVADSTYWTFGPGGPRKCEREPAGWSDTGWVC 176 FL-246WCSMCSVLRAFNCPYFCPWL 177 FL-247 PWIFDRSVVCEEREAPRRHL 178 FL-251TSWRHRMPTGTDRCCFLVQL 179 FL-252 RPWSSDTSVWWDGLFGMNYS 180 FL-254RPWSSDTSVWWDGLFGMNYS 181 FL-256 LVTALIMSESIVRMNPMYLT 182 FL-257LLACSVYWLWQRPCDGCLFM 183 FL-258 GVNNGGTNDPEGVSEASWIP 184 FL-262LLACSVYWLWQRPCDGCLFM 185 FL-265 GIVSKQGADEGMLEIFASSW 186 FL-275TIDQRMVSLGAIWWSYPRCW 187 FL-278 PPWTKRPLTSGGVRGELWVW 188 FL-280RVDSLGIKLDKSTLVTVHVV 189 FL-281 TEACQVVLLGKRSLLPVVAG 190 FL-292AQRSCWERLWTGQWRRLPLM 191 FL-293 AQRSCWERLWTGQWRRSASD 192 FL-294VSWSAAGRGYVFMYRWSPRC 193 FL-300 FGVIVTNAASEFTTRGYDSC 194 FL-302SFSSLFLAWLMQTGQEAGTV 195 FL-304 SRSDLWVSWCRNLLDGQSWS 196 FL-312HPYLITDIISMYRSPWSVPA 197 FL-313 RDALTNIGRSICALLLVLCK 198 FL-322WGYFGSFVGGVFDVWFSGVA 199 FL-325 SRS DLWVSWCRNLLDGQSWS 200 FL-326RNGRNICVLSVCSRFSHFNP 201 FL-328 GGSDERYFWYQSFSSCAYEW 202 FL-334GVLFTFKKYPQGLSCTTSYG 203 FL-341 LLACSVYWLWQRPCDGCLFM 204 FL-352VVQNVRGWLVYCCADFHTYV 205 FL-355 LRGVSPWLQSFVSIAVQSCK 206 FL-364TGSGLTPCLHCRVQFQRSYL 207 FL-365 TRMHSLCSGFCVICMGGPRV 208 FL-380RNGRNICVLSVCSRFSHFNP 209 FL-381 MLGIWRVLHEMVVPLKLGVD 210 FL-382CPDCPLSSVLRTATTAFFGG 211 FL-434 RGRMEADKSFDSTCLRCGCS

The peptide aptamers of the present invention were isolated in astringent in vivo screen and, as such, are likely to bind to viralreplication initiation proteins with high affinity, fold correctly andbe stably expressed in a cellular environment. Some of the peptideaptamers were selected for binding to the N-terminus of AL1, which doesnot resemble plant proteins. Consequently, the peptide aptamers areunlikely to interact with host proteins, minimizing the risk that theirexpression will be toxic to plants. Further, the peptide aptamers areca. 12 kD—a size typical of a small, stable protein that can movepassively into the nucleus where replication proteins are localized.Additionally, several of the aptamer peptides reduced TGMV DNAaccumulation more strongly than a trans-dominant negative TAL1 mutantthat can confer immunity to TGMV infection when expressed in transgenicplants. Finally, the ability of the aptamer peptides to bind to thedivergent TAL1 and CaAL1 proteins suggests that these peptides recognizeconserved features in the N-termini of geminivirus replication proteins.This observation represents a key difference from interferencestrategies based on viral sequences like trans-dominant negativemutants, antisense RNAs and RNAi constructs, all of which are onlyeffective against the cognate geminivirus or closely related viruses. Incontrast, a resistance strategy based on the interfering aptamerpeptides could be broadly applicable to all geminivirus genera and othereukaryotic single-stranded DNA viruses with related replication proteinsand could confer resistance to mixed infections and viral variants.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is describedby the following claims, with equivalents of the claims to be includedtherein.

1. A polypeptide comprising an amino acid sequence selected from thegroup consisting of: (a) the amino acid sequence of SEQ ID NO:38; (b) afragment of the amino acid sequence of SEQ ID NO:38, wherein thefragment comprises MHXXXXXG; and wherein the fragment binds to a viralreplication (Rep) protein; and (c) an amino acid sequence that is atleast 80% similar to the amino acid sequence of (a) or (b) and binds toa viral Rep protein.
 2. The polypeptide according to claim 1, whereinthe polypeptide binds to a Rep protein selected from the groupconsisting of a geminivirus Rep protein, a nanovirus Rep protein, acircovirus Rep protein and combinations thereof.
 3. The polypeptideaccording to claim 1, wherein the polypeptide binds to a geminivirus Repprotein.
 4. The polypeptide according to claim 1, wherein thepolypeptide binds to a Rep protein selected from the group consisting ofa tomato golden mosaic virus (TGMV) Rep protein, a cabbage leaf curlvirus (CbLCV) Rep protein, a tomato yellow leaf curl virus (TYLCV) Repprotein, a tomato mottle virus (ToMoV) Rep protein, an African cassavamosaic virus (ACMV) Rep protein, a maize streak virus (MSV) Rep protein,and a cotton leaf curl virus (CLCuV) Rep protein, and combinationsthereof.
 5. The polypeptide according to claim 1, wherein thepolypeptide comprises an amino acid sequence that is at least 95%similar to the amino acid sequence of (a) and binds to a viral Repprotein.
 6. The polypeptide according to claim 5, wherein the amino acidsequence comprises MHXXXXXG.
 7. The polypeptide according to claim 1consisting of the amino acid sequence of SEQ ID NO:38.
 8. A fusionprotein comprising the polypeptide according to claim
 1. 9. The fusionprotein according to claim 8, wherein the fusion protein comprisesthioredoxin.
 10. The polypeptide according to claim 1, wherein thepolypeptide comprises a fragment of the amino acid sequence of SEQ IDNO:38, wherein the fragment comprises MHXXXXXG, and wherein the fragmentbinds to a viral Rep protein.
 11. The polypeptide according to claim 1,wherein the amino acid sequence comprises no more than three amino acidsubstitutions, insertions and/or deletions as compared with the aminoacid sequence of (a).
 12. The polypeptide according to claim 1, whereinthe amino acid sequence comprises no more than two amino acidsubstitutions as compared with the amino acid sequence of (a), andwherein all of the amino acid substitutions are conservativesubstitutions.
 13. The polypeptide according to claim 1, wherein thepolypeptide inhibits viral replication.
 14. The polypeptide according toclaim 13, wherein the polypeptide inhibits geminivirus, nanovirus and/orcircovirus replication.
 15. The polypeptide according to claim 13,wherein the polypeptide inhibits geminivirus replication.
 16. Thepolypeptide according to claim 15, wherein the polypeptide inhibitstomato golden mosaic virus (TGMV), tomato yellow leaf curl virus (TYLCV)replication, cabbage leaf curl virus (CbLCV) replication, tomato mottlevirus (ToMoV) replication, African cassava mosaic virus (ACMV)replication, maize streak virus (MSV) replication and/or cotton leafcurl virus (CLCuV) replication.
 17. The polypeptide according to claim1, wherein the polypeptide inhibits viral infection.
 18. The polypeptideaccording to claim 17, wherein the polypeptide inhibits geminivirus,nanovirus and/or circovirus infection.
 19. The polypeptide according toclaim 17, wherein the polypeptide inhibits geminivirus replication. 20.The polypeptide according to claim 19, wherein the polypeptide inhibitstomato golden mosaic virus (TGMV), tomato yellow leaf curl virus (TYLCV)infection, cabbage leaf curl virus (CbLCV) infection, tomato mottlevirus (ToMoV) infection, African cassava mosaic virus (ACMV) infection,maize streak virus (MSV) infection and/or cotton leaf curl virus (CLCuV)infection.
 21. A method of detecting a viral infection, the methodcomprising: (a) contacting a sample with a polypeptide according toclaim 1; and (b) detecting the presence or absence of binding betweenthe polypeptide and a target, wherein the binding of the polypeptide tothe target in the sample indicates the presence of a virus.
 22. Themethod of claim 21, wherein the target is a viral Rep protein.
 23. Amethod of detecting a viral infection, the method comprising: (a)contacting a sample with a fusion protein according to claim 8; and (b)detecting the presence or absence of binding between the polypeptide anda target, wherein the binding of the polypeptide to the target in thesample indicates the presence of a virus.
 24. A method of inhibitingviral replication in a plant cell, the method comprising introducing thepolypeptide of claim 1 into the plant cell in an amount effective toinhibit viral replication.
 25. A method of inhibiting viral replicationin a plant cell, the method comprising introducing the fusion protein ofclaim 8 into the plant cell in an amount effective to inhibit viralreplication.
 26. A method of providing increased resistance to viralinfection in a plant, the method comprising introducing the polypeptideof claim 1 into the plant in an amount effective to increase resistanceto viral infection.
 27. A method of providing increased resistance toviral infection in a plant, the method comprising introducing the fusionprotein of claim 8 into the plant in an amount effective to increaseresistance to viral infection.